Amphiphilic entity nanoparticles

ABSTRACT

The present invention provides nanoparticle compositions comprising AE nanoparticles. The present invention provides AE nanoparticles comprising one or more amphiphilic entities and pharmaceutical compositions comprising AE nanoparticles. The present invention provides methods of manufacturing AE nanoparticles. The present invention provides methods of delivering a biologically active agent to a subject by administering AE nanoparticles containing a biologically active agent to a subject.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/379,364, filed Apr. 9, 2019, which, in turn, is a continuation ofU.S. patent application Ser. No. 15/636,927, filed Jun. 29, 2017, nowU.S. Pat. No. 10,285,941, issued May 14, 2019, which, in turn, is acontinuation of U.S. patent application Ser. No. 14/182,919, filed Feb.18, 2014, now U.S. Pat. No. 9,724,299, issued Aug. 8, 2017, which is acontinuation of U.S. patent application Ser. No. 12/517,154, filed Feb.24, 2010, which is a 35 U.S.C. § 371 National Stage of InternationalApplication No. PCT/US07/86018, filed Nov. 30, 2007, which claimspriority under 35 U.S.C. § 119(e) to U.S. provisional patentapplication, U.S. Ser. No. 60/872,198, filed Dec. 1, 2006. The entirecontents of each of the foregoing applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

A significant literature exists on strategies for producing polymernanoparticles. For example, when an amphiphilic polymer is present in asolvent at a concentration above its critical micellar concentration, itwill self-assemble into nanoparticle structures. Even when such apolymer is present at a lower concentration, it can be caused to formnanoparticles by decreasing the solvation of the solvent such as bydiluting the solvent with water. Solvents that have traditionally beenused to manufacture nanoparticles were usually one or more of dimethylsulfoxide (DMSO), dimethyl acetimide, dimethyl formamide, chloroform,tetramethyl formamide. Unfortunately, each of these is highly toxic andexpensive.

In addition to the fact that they require one or more toxic solvents,standard methods for generating polymer nanoparticles tend to be slow(requiring up to several days to complete nanoparticle assembly), and togive only low yields of nanoparticles. Thus, the cost of the components,the speed and yield of the chemical reaction, the toxicity of theresidual components, and the overall expense of the available processesfor producing nanoparticles can have a profound negative impact on thecommercial feasibility of using such nanoparticles.

Nanoparticles are often proposed for use as a delivery mechanism for abiologically active agent, such as a pharmaceutical. Frequently, it hasbeen challenging to incorporate (or “load”) the biologically activeagent into the nanoparticle because the amount that can be incorporatedis limited or that it takes a great deal of time to incorporate thematerial (through, for example, diffusion). This challenge can limit thepractical or commercial utility of the nanoparticle as a deliverymechanism for a biologically active agent.

Therefore, there is a need for the development of inexpensive, efficientmethods of manufacturing nanoparticles. There is a further need for thedevelopment of methods of manufacturing nanoparticles that do notutilize or leave behind toxic residual components or reactionby-products.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides nanoparticlecompositions comprising amphiphilic entity (AE) nanoparticles. In someembodiments, the nanoparticle compositions are uniform. For example, insome embodiments, the total particle distribution is encompassed withina particular range of particle diameter sizes (discussed in more detailbelow); in some embodiments, a portion of the total particledistribution is outside of the particular range.

In some embodiments, the present invention provides AE nanoparticles andpharmaceutical compositions comprising AE nanoparticles. In someembodiments, AE nanoparticles comprise one or more amphiphilic entities.The AE nanoparticles may comprise one or more dispersion media,surfactants, biologically active agents, and/or release-retardingingredients. In some embodiments, the AE nanoparticles are smaller than1000 nanometers, 500 nanometers, 200 nanometers, or 100 nanometers indiameter.

In some embodiments, the present invention provides systems, includingmethods, reagents, and/or compositions, for manufacturing AEnanoparticles. In some embodiments, AE nanoparticle compositions may begenerated by exposure to high shear forces; in some embodiments, AEnanoparticle compositions may be generated high pressure homogenization;in some embodiments, AE nanoparticle compositions may be generated bycavitation; in some embodiments, AE nanoparticle compositions may begenerated by microfluidization. In some embodiments, commerciallyavailable equipment, such the Microfluizider®, may be used to generatehigh shear forces.

In some embodiments, methods of manufacturing AE nanoparticles generallycomprise steps of preparing a premix of one or more amphiphilic entitiesand applying high shear force to the premix. The premix generallycontains one or ore amphiphilic entities and one ore more dispersionmedia. The premix may optionally comprise one or more surfactants,biologically active agents, and/or release-retarding ingredients. Insome embodiments, the amphiphilic entities of the premix assemble intoparticles (e.g. nanoparticles, microparticles, and/or micelles) beforeapplication of high shear force. In some embodiments, the amphiphilicentities of the premix do not assemble into particles before theapplication of high shear force.

In some embodiments, the present invention provides improvements overtraditional methods of manufacturing nanoparticles. For example, the useof mechanical energy replaces or minimizes the requirement to use costlyand toxic chemical solvents, increases the speed and reaction yield,reduces the overall cost of the synthetic reaction, thereby increasingthe commercial utility of AE nanoparticles. Additionally, the use ofhigh shear force allows for increased loading capacity of thenanoparticle as compared to traditional methods of formingnanoparticles. In traditional methods, loading of agents within or onthe surface of nanoparticles relies on diffusion of the agent to theinterior and/or to the surface of the nanoparticle.

The present invention encompasses the recognition that subjectingparticles (e.g. nanoparticles, microparticles, and/or micelles) to highshear force is a method of manufacturing nanoparticles that isinexpensive and efficient and does not utilize toxic residualcomponents. In some embodiments, the AE nanoparticles are completelyfree or substantially free of toxic components. In some embodiments, thenanoparticle compositions comprising AE nanoparticles are completelyfree or substantially free of toxic components. The present inventionfurther encompasses the recognition that subjecting particles (e.g.nanoparticles, microparticles, and/or micelles) to high shear forcegenerates nanoparticles with an increased loading capacity relative totraditional methods of making nanoparticles.

In some embodiments, the present invention provides a method ofdelivering a composition, substance, or biologically active agent to asubject. In some embodiments, the composition, substance, orbiologically active agent may be delivered via any route. In someembodiments, the composition, substance, or biologically active agent isdelivered transdermally (or topically).

In some embodiments, the invention provides methods and compositions fortransdermally delivering a biologically active agent to a subject byadministering to the subject nanoparticle compositions comprising AEnanoparticles to the surface of the subject's skin, wherein thebiologically active agent is encapsulated within and/or bound to thesurface of the AE nanoparticles. The present invention encompasses thediscovery that AE nanoparticles of the present invention can achievetransdermal delivery of a biologically active agent without changing oraltering the structure of the skin. For example, abrasive agents oragents that erode the top layer of the skin (whether chemical,mechanical, electrical, magnetic, etc.) are not required to achievetransdermal delivery of a biologically active agent. In someembodiments, a composition for transdermal delivery of a biologicallyactive agent may be in the form of a transdermal patch. In someembodiments, inventive compositions comprising AE nanoparticles fortransdermal delivery of a biologically active agent may be used in anapplication device that permits application of the composition to atarget site on the skin without applying the composition to non-targetsite areas of the skin.

In some embodiments, the biologically active agent comprises abiological polymer. In some embodiments, the polymer is DNA, RNA, or aprotein. In some embodiments, the protein comprises multiple proteinsand/or protein complexes. In certain embodiments, the biologicallyactive agent delivered according to the present invention is one or morebotulinum toxin peptides, polypeptides and/or protein complexes. In someembodiments, the botulinum toxin may be one or more of botulinum toxinserotypes A, B, C₁, C₂, D, E, F, or G. In some embodiments, thebotulinum toxin may be an isolated and/or purified botulinum toxin. Insome embodiments, the botulinum toxin may be a partially-isolated and/orpartially-purified botulinum toxin. In some embodiments, the botulinumtoxin may be a native botulinum complex. In some embodiments, thebotulinum toxin may be associated with non-toxin proteins. In someembodiments, the botulinum toxin may be a recombinantly-made botulinumtoxin.

In some embodiments, the present invention provides methods of treatingfacial wrinkles (e.g. wrinkles involving the forehead, glabellar,rhytids, and/or periorbital regions); hyperkinetic facial lines;platysma bands; neuromuscular disorders and conditions involvingmuscular spasm and/or contracture (e.g. facial palsy, blepharospasm,cerebral palsy, strabismus, and/or dystonia); prostate hyperplasia;hyperhidrosis; headache, and/or temporomandibular joint diseases anddisorders (TMJ, also known as “lockjaw”). Such methods generally involveadministering to a subject nanoparticle compositions comprising AEnanoparticles for the transdermal delivery of a botulinum toxin orbotulinum toxin complex.

In some embodiments, the present invention provides methods of treatingfacial wrinkles (e.g. wrinkles involving the forehead, glabellar,rhytids, and/or periorbital regions), hyperkinetic facial lines, and/orplatysma bands. In some embodiments, nanoparticle compositionscomprising AE nanoparticles for the transdermal delivery of abiologically active agent may be used to treat facial wrinkles. In someembodiments, nanoparticle compositions comprising AE nanoparticles forthe transdermal delivery of a botulinum toxin or botulinum toxin complexmay be used to treat facial wrinkles. In some embodiments, facialwrinkles may include glabellar wrinkles, facial lines (e.g. hyperkineticfacial lines), forehead frown lines, midfacial wrinkles, mouth wrinkles,neck lines and banding (e.g. platysma bands), and chin creases. Suchmethods generally involve administering to a subject nanoparticlecompositions comprising AE nanoparticles for the transdermal delivery ofa botulinum toxin or botulinum toxin complex.

In some embodiments, the present invention does not provide methods oftreating prostate hyperplasia. In some embodiments, the presentinvention does not provide methods of treating neuromuscular disordersand conditions involving muscular spasm and/or contracture. In someembodiments, the present invention does not provide methods of treatinghyperhidrosis. In some embodiments, the present invention does notprovide methods of treating headache. In some embodiments, the presentinvention does not provide methods of treating TMJ.

This application refers to various patent publications, all of which areincorporated herein by reference.

DEFINITIONS

Abrasion: The term “abrasion,” as used herein refers to any means ofaltering, disrupting, removing, or destroying the top layer of the skin.In some embodiments, abrasion refers to a mechanical means of altering,disrupting, removing, or destroying the top layer of the skin. In someembodiments, abrasion refers to a chemical means of altering,disrupting, removing, or destroying the top layer of skin. To give but afew examples, agents such as exfoliants, fine particles (e.g. magnesiumor aluminum particles), acids (e.g. alpha-hydroxy acids or beta-hydroxyacids), alcohols, may cause abrasion. In general, permeation enhancerssuch as those described, for example, by Donovan (e.g. U.S. PatentPublications 2004/009180 and 2005/175636 and PCT Publication WO04/06954; all of which are incorporated herein by reference), and Graham(e.g. U.S. Pat. No. 6,939,852 and U.S. Patent Publication 2006/093624;both of which are incorporated herein by reference), etc., are expectedto cause abrasion. Of course, those of ordinary skill in the art willappreciate that a particular agent may cause abrasion when present atone concentration, or in association with one or more other agents, butmay not cause abrasion under different circumstances. Thus, whether ornot a particular material is an “abrasive agent” depends on context.Abrasion can readily be assessed by those of ordinary skill in the art,for example by observation of redness or irritation of the skin and/orhistologic examination of skin showing alteration, disruption, removal,or erosion of the stratum corneum.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally-occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a D-aminoacid; in some embodiments, an amino acid is an L-amino acid. “Standardamino acid” refers to any of the twenty standard L-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, and/or substitution with otherchemical groups that can change the peptide's circulating half-lifewithout adversely affecting their activity. Amino acids may participatein a disulfide bond. The term “amino acid” is used interchangeably with“amino acid residue,” and may refer to a free amino acid and/or to anamino acid residue of a peptide. It will be apparent from the context inwhich the term is used whether it refers to a free amino acid or aresidue of a peptide.

Amphiphilic entity: As used herein, the term “amphiphilic entity” refersto a chemical entity possessing both hydrophilic and hydrophobic nature.As used herein, the terms “amphiphilic” and “amphipathic” can be usedinterchangeably. In some embodiments, the amphiphilic entities of an AEnanoparticle are biocompatible. Biocompatible amphiphilic entities arenot significantly toxic to cells. In some embodiments, the amphiphilicentities of an AE nanoparticle are biodegradable. Biodegradableamphiphilic entities are broken down by the cellular machinery and/or byhydrolysis into components that the cells can either reuse or dispose ofwithout significant toxic effect. In some embodiments, a biodegradableamphiphilic entity and its biodegradation byproducts are biocompatible.In some embodiments, the amphiphilic entity is non-immunogenic. In someembodiments, an amphiphilic entity may comprise one or more individualcompounds or molecules that is itself amphiphilic. In some embodiments,an amphiphilic entity may comprise one or more individual componentsthat is not itself amphiphilic but that has some hydrophilic orhydrophobic character. Typically, the individual components areassociated with one another such that the assemblage of the individualcomponents is amphiphilic.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, genetically-engineered animal, and/ora clone.

Antibody: As used herein, the term “antibody” refers to animmunoglobulin, whether naturally produced, synthetically produced, orboth. The antibody may be a member of any immunoglobulin class,including any of the human classes: IgG, IgM, IgA, IgD, and/or IgE. Theantibody may be a fragment of an antibody such as an Fab′; F(ab′)₂; scFv(single-chain variable) and/or any other fragment that retains anantigen binding site; and/or a recombinantly-produced scFv fragment,including recombinantly-produced fragments (see, e.g., Allen, 2002, NatRev Cancer, 2:750 and references therein; incorporated herein byreference). In certain embodiments of the invention the term refers to“humanized” antibodies, which include sequences of human origin. In someembodiments, “humanized” antibodies are characterized by a variabledomain of rodent origin fused to a constant domain of human origin, thusretaining the specificity of the rodent antibody. It is noted that thedomain of human origin need not originate directly from a human in thesense that it is first synthesized in a human being. For example,“human” domains may be generated in rodents whose genome incorporateshuman immunoglobulin genes (see, e.g., Harlow et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988; incorporated herein by reference). An antibody maybe polyclonal or monoclonal.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active agent: As used herein, the phrase “biologicallyactive agent” refers to any substance that has activity in a biologicalsystem and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments,where a protein or polypeptide is biologically active, a portion of thatprotein or polypeptide that shares at least one biological activity ofthe protein or polypeptide is typically referred to as a “biologicallyactive” portion.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a substance, in the broadest sense, is one that shares somedegree of sequence and/or structural identity and/or at least onefunctional characteristic with the relevant intact substance. Forexample, a “characteristic portion” of a protein or polypeptide is onethat contains a continuous stretch of amino acids, or a collection ofcontinuous stretches of amino acids, that together are characteristic ofa protein or polypeptide. In some embodiments, each such continuousstretch generally will contain at least 2, 5, 10, 15, 20 or more aminoacids. In general, a characteristic portion is one that, in addition tothe sequence identity specified above, shares at least one functionalcharacteristic with the relevant intact protein. In some embodiments,the characteristic portion may be biologically active.

Dispersion medium: The term “dispersion medium,” as used herein, refersto a liquid medium in which particles (e.g., AE nanoparticles) aredispersed. In general, a dispersion is formed when at least twoimmiscible materials are combined. An “oil-in-water” dispersion is onein which oily particles are dispersed within an aqueous dispersionmedium. A “water-in-oil” dispersion is one in which aqueous particlesare dispersed within an oily dispersion medium. Those of ordinary skillin the art will appreciate that a dispersion can be formed from any twoimmiscible media and is not limited strictly to combinations of aqueousand oily media. The term “dispersion medium” therefore applies broadlyto any dispersion medium notwithstanding that it is common to refer to“aqueous” and “oily” categories.

Encapsulated: The term “encapsulated” (also “encapsulate” or“encapsulating”) is used herein to mean that the encapsulated entity iscompletely surrounded by another material. To give but one example, abiologically active agent may be encapsulated within a nanoparticle inan inventive composition. Such encapsulation may be achieved, forexample, during formation of a nanoparticle composition, for exampleduring exposure to high shear force.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end formation); (3) translation of an RNA into a polypeptide orprotein; (4) post-translational modification of a polypeptide orprotein.

Gene: As used herein, the term “gene” has its meaning as understood inthe art. It will be appreciated by those of ordinary skill in the artthat the term “gene” may include gene regulatory sequences (e.g.,promoters, enhancers, etc.) and/or intron sequences. It will further beappreciated that definitions of gene include references to nucleic acidsthat do not encode proteins but rather encode functional RNA moleculessuch as tRNAs. For the purpose of clarity we note that, as used in thepresent application, the term “gene” generally refers to a portion of anucleic acid that encodes a protein; the term may optionally encompassregulatory sequences, as will be clear from context to those of ordinaryskill in the art. This definition is not intended to exclude applicationof the term “gene” to non-protein-coding expression units but rather toclarify that, in most cases, the term as used in this document refers toa protein-coding nucleic acid.

Gene product or expression product: As used herein, the term “geneproduct” or “expression product” generally refers to an RNA transcribedfrom the gene (pre-and/or post-processing) or a polypeptide (pre- and/orpost-modification) encoded by an RNA transcribed from the gene.

Homology: As used herein, the terms “homology” and “identity” are usedinterchangeably and refer to the overall relatedness between polymericmolecules, e.g. between nucleic acid molecules (e.g. DNA moleculesand/or RNA molecules) and/or between polypeptide molecules. Calculationof the percent homology or identity of two nucleic acid sequences can beperformed by aligning the two sequences for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondnucleic acid sequences for optimal alignment and non-homologoussequences can be disregarded for comparison purposes). In certainembodiments, the length of a sequence aligned for comparison purposes isat least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or 100% of the length of thereference sequence. Nucleotides at corresponding nucleotide positionsare then compared. When a position in the first sequence is occupied bythe same nucleotide as the corresponding position in the secondsequence, then the molecules are identical (or homologous) at thatposition. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences, takinginto account the number of gaps, and the length of each gap, which needsto be introduced for optimal alignment of the two sequences. Thecomparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm. Forexample, the percent identity between two nucleotide sequences can bedetermined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17; incorporated herein by reference), which has been incorporatedinto the ALIGN program (version 2.0) using a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4. The percentidentity between two nucleotide sequences can, alternatively, bedetermined using the GAP program in the GCG software package using anNWSgapdna.CMP matrix.

Hydrophilic: As used herein, a “hydrophilic” substance is a substancethat may be soluble in polar dispersion media. In some embodiments, ahydrophilic substance can transiently bond with polar dispersion media.In some embodiments, a hydrophilic substance transiently bonds withpolar dispersion media through hydrogen bonding. In some embodiments,the polar dispersion medium is water. In some embodiments, a hydrophilicsubstance may be ionic. In some embodiments, a hydrophilic substance maybe non-ionic. In some embodiments, a hydrophilic substance may dissolvemore readily in water, polar dispersion media, or hydrophilic dispersionmedia than in oil, non-polar dispersion media, or hydrophobic dispersionmedia. In some embodiments, a hydrophilic substance may dissolve lessreadily in oil, non-polar dispersion media, or hydrophobic dispersionmedia than in water, polar dispersion media, or hydrophilic dispersionmedia. In some embodiments, a substance is hydrophilic relative toanother substance because it is more soluble in water, polar dispersionmedia, or hydrophilic dispersion media than is the other substance. Insome embodiments, a substance is hydrophilic relative to anothersubstance because it is less soluble in oil, non-polar dispersion media,or hydrophobic dispersion media than is the other substance.

Hydrophobic: As used herein, a “hydrophobic” substance is a substancethat may be soluble in non-polar dispersion media. In some embodiments,a hydrophobic substance is repelled from polar dispersion media. In someembodiments, the polar dispersion medium is water. In some embodiments,hydrophobic substances are non-polar. In some embodiments, a hydrophobicsubstance may dissolve more readily in oil, non-polar dispersion media,or hydrophobic dispersion media than in water, polar dispersion media,or hydrophilic dispersion media. In some embodiments, a hydrophobicsubstance may dissolve less readily in water, polar dispersion media, orhydrophilic dispersion media than in oil, non-polar dispersion media, orhydrophobic dispersion media. In some embodiments, a substance ishydrophobic relative to another substance because it is more soluble inoil, non-polar dispersion media, or hydrophobic dispersion media than isthe other substance. In some embodiments, a substance is hydrophobicrelative to another substance because it is less soluble in water, polardispersion media, or hydrophilic dispersion media than is the othersubstance.

In conjunction with: As used herein, the phrase “delivered inconjunction with” refers to the co-delivery of two or more substances oragents. In particular, according to the present invention, the phrase isused herein in reference to delivery of a biologically active agent withinventive AE nanoparticles and/or nanoparticle compositions. A substanceor agent is delivered in conjunction with AE nanoparticles when thesubstance or agent is combined with AE nanoparticles and/or nanoparticlecompositions; is encapsulated or completely surrounded by AEnanoparticles; is embedded within an AE nanoparticle micellar membrane;and/or is associated with the outer surface of an AE nanoparticlemicellar membrane. A substance or agent to be delivered in conjunctionwith AE nanoparticles and/or nanoparticle compositions may or may not becovalently linked to the AE nanoparticles and/or nanoparticlecompositions. A substance or agent to be delivered in conjunction withnanoparticles and/or nanoparticle compositions may or may not beattached to the AE nanoparticles and/or nanoparticle compositions byadsorption forces.

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or more of the other components with which they were initiallyassociated. In some embodiments, isolated substances and/or entities aremore than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.

Microfluidized: As used herein, the term “microfluidized” means exposedto high shear forces. In some embodiments, such exposure to high shearforces is accomplished by exposure to high pressure; in some embodimentssuch high pressure is within the range of about 15,000 to about 26,000psi. In some embodiments, such exposure to high shear forces isaccomplished by cavitation. In some embodiments, such exposure to highshear forces is accomplished by passing a sample through an instrumentsuch as, for example, a Microfluidizer® (Microfluidics Corporation/MFICCorporation) or other like device that may be useful in creating auniform nanoparticle composition. In some embodiments of the presentinvention, a sample is microfluidized through exposure to high shearforces for a period of time less than about 10 minutes. In someembodiments, the period of time is less than about 9, 8, 7, 6, 5, 4, 3,2, or 1 minute(s). In some embodiments, the period of time is within therange of about 1-2 minutes. In some embodiments, the period of time isless than 1 minute. In some embodiments, the period of time is about 30seconds. In some embodiments of the invention, a sample is“microfluidized” through a single exposure to high shear forces; suchembodiments are referred to as “single pass” microfluidization.

Nanoparticle: As used herein, the term “nanoparticle” refers to anyparticle having a diameter of less than 1000 nanometers (nm). In someembodiments, a nanoparticle has a diameter of less than 300 nm, asdefined by the National Science Foundation. In some embodiments, ananoparticle has a diameter of less than 100 nm as defined by theNational Institutes of Health. In some embodiments, nanoparticles aremicelles in that they comprise an enclosed compartment, separated fromthe bulk solution by a micellar membrane. A “micellar membrane”comprises amphiphilic entities which have aggregated to surround andenclose a space or compartment (e.g., to define a lumen).

Nanoparticle composition: As used herein, the term “nanoparticlecomposition” refers to any substance that contains at least one AEnanoparticle. In some embodiments, a nanoparticle composition is auniform collection of AE nanoparticles. In some embodiments,nanoparticle compositions are dispersions or emulsions. In general, adispersion or emulsion is formed when at least two immiscible materialsare combined. An “oil-in-water” dispersion is one in which oilyparticles (or hydrophobic or non-polar) are dispersed within an aqueousdispersion medium. A “water-in-oil” dispersion is one in which aqueous(or hydrophilic or polar) particles are dispersed within an oilydispersion medium. Those of ordinary skill in the art will appreciatethat a dispersion can be formed from any two immiscible media and is notlimited strictly to combinations of aqueous and oily media. The term“dispersion medium” therefore applies broadly to any dispersion mediumnotwithstanding that it is common to refer to “aqueous” and “oily”categories. In some embodiments, nanoparticle compositions arenanoemulsions. In some embodiments, nanoparticle compositions comprisemicelles. In some embodiments, a nanoparticle composition is stable. Insome embodiments, a nanoparticle composition includes one or morebiologically active agents to be delivered in conjunction with the AEnanoparticles.

Not contaminated with: The phrase “not contaminated with,” when usedherein to refer to a nanoparticle composition, is synonymous with“substantially free of” and describes a nanoparticle compositioncontaining no more than about 50% of the recited material. For example,if a nanoparticle composition is said to be “substantially free of”particles whose diameter is outside of a stated range, then no more thanabout 50% of the particles in that composition have diameters outside ofthe range. In some embodiments, no more than 25% of the particles areoutside of the range. In some embodiments, no more than 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5% or less of the particles have diameters outside of the statedrange.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that can be incorporatedinto an oligonucleotide chain. In some embodiments, “nucleic acid”encompasses RNA as well as single and/or double-stranded DNA and/orcDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/orsimilar terms include nucleic acid analogs, i.e. analogs having otherthan a phosphodiester backbone. For example, the so-called “peptidenucleic acids,” which are known in the art and have peptide bondsinstead of phosphodiester bonds in the backbone, are considered withinthe scope of the present invention. The term “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and/or encode the same amino acidsequence. Nucleotide sequences that encode proteins and/or RNA mayinclude introns.

Nutraceutical: As used herein, the term “nutraceutical” refers to anysubstance thought to provide medical, health, or biological benefits. Insome embodiments, nutraceuticals may prevent disease. In someembodiments, nutraceuticals may provide basic nutritional value. In someembodiments, a nutraceutical is a food or part of a food. In someembodiments, a nutraceutical agent may be a class of isolated nutrients,dietary supplements, vitamins, minerals, herbs, fortified foods, healingfoods, genetically engineered foods, and processed foods. Nutraceuticalsmay also be known as “phytochemical foods” or “functional foods.”

Patient: A “patient,” or “subject,” as used herein, means an animal. Insome embodiments, the animal is a mammal, commonly a human.

Premix: As used herein, the term “premix” refers to any combination ofcomponents that is subsequently used to generate a nanoparticlecomposition according to the present invention. For example, a premix isany collection of ingredients that, when subjected to high shear forces,generates AE nanoparticles according to the present invention. In someembodiments, a premix contains two or more immiscible solvents. In someembodiments, a premix contains components that self-assemble intomicroparticles or nanoparticles. In some embodiments, a premix containscomponents that self-assemble into micelles. In some embodiments, apremix contains one or more peptides as described in PCT applicationserial number PCT/US07/086040 entitled “Peptide Nanoparticles and UsesTherefor,” filed Nov. 30, 2007. In some embodiments, a premix containsone or more unmodified peptides; in some embodiments, a premix containsat least one other biologically active agent. In some embodiments, apremix is agitated, mixed, and/or stirred; in some embodiments, a premixis agitated, mixed, and/or stirred prior to being subjected to highshear force. In some embodiments, a premix comprises at least onesolubilized component (i.e., at least one component that is insolution); in some such embodiments, the premix is subjected to highshear force after such solubilization is achieved.

Pure: As used herein, a substance and/or entity is “pure” if it issubstantially free of other components. For example, a preparation thatcontains more than about 90% of a particular substance and/or entity istypically considered to be a pure preparation. In some embodiments, asubstance and/or entity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% pure.

Shear force: As used herein, the term “shear force” refers to a forcethat is parallel to the face of a material, as opposed to a force thatis perpendicular to the face of a material. In some embodiments, acomposition exposed to high shear forces in order to produce a uniformnanoparticle composition. Any method known in the art can be used togenerate high shear forces. In some embodiments, cavitation is used togenerate high shear forces. In some embodiments, high pressurehomogenization is used to generate high shear forces. Alternatively oradditionally, high shear force may be administered by exposure to highpressure, for example about 15,000 psi. In some embodiments, such highpressure is within the range of about 18,000 to about 26,000 psi; insome embodiments, it is within the range of about 20,000 to about 25,000psi. In some embodiments, a Microfluidizer® Processor (MicrofluidicsCorporation/MFIC Corporation) or other like device is used to generatehigh shear force. Microfluidizer® Processors provide high pressure and aresultant high shear rate by accelerating a composition throughmicrochannels (typically having dimensions on the order of 75 microns)at a high velocity (typically in the range of 50 m/s to 300 m/s) forsize reduction to the nanoscale range. As the fluid exits themicrochannels it forms jets which collide with jets from opposingmicrochannels. In the channels the fluid experiences high shear (up to10⁷ 1/s) which is orders of magnitude higher than that of conventionaltechnologies. Jet collisions result in mixing in submicron level.Therefore, in such devices, high shear and/or impact can achieveparticle size reduction and mixing of multiphase. In some embodiments ofthe present invention, a sample is exposed to high shear forces for aperiod of time less than about 10 minutes. In some embodiments, theperiod of time is less than about 9, about 8, about 7, about 6, about 5,about 4, about 3, about 2, or about 1 minute(s). In some embodiments,the period of time is within the range of about 1 to about 2 minutes orless; in some embodiments, the period of time is about 30 seconds. Insome embodiments of the invention, a sample is “microfluidized” througha single exposure to high shear forces; such embodiments are referred toherein as “single pass” microfluidization.

Small Molecule: In general, a “small molecule” is understood in the artto be an organic molecule that is less than about 5 kilodaltons (Kd) insize. In some embodiments, the small molecule is less than about 3 Kd,about 2 Kd, or about 1 Kd. In some embodiments, the small molecule isless than about 800 daltons (D), about 600 D, about 500 D, about 400 D,about 300 D, about 200 D, or about 100 D. In some embodiments, smallmolecules are non-polymeric. In some embodiments, small molecules arenot proteins, peptides, or amino acids. In some embodiments, smallmolecules are not nucleic acids or nucleotides. In some embodiments,small molecules are not saccharides or polysaccharides.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition of this invention may be administered,e.g., for experimental, diagnostic, prophylactic, and/or therapeuticpurposes. Typical subjects include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and humans; insects; worms; etc.).

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Stable: The term “stable,” when applied to nanoparticle compositionsherein, means that the compositions maintain one or more aspects oftheir physical structure (e.g., size range and/or distribution ofparticles) over a period of time. In some embodiments of the invention,a stable nanoparticle composition is one for which the average particlesize, the maximum particle size, the range of particle sizes, and/or thedistribution of particle sizes (i.e., the percentage of particles abovea designated size and/or outside a designated range of sizes) ismaintained for a period of time. In some embodiments, the period of timeis at least about one hour; in some embodiments the period of time isabout 5 hours, about 10 hours, about one (1) day, about one (1) week,about two (2) weeks, about one (1) month, about two (2) months, aboutthree (3) months, about four (4) months, about five (5) months, aboutsix (6) months, about eight (8) months, about ten (10) months, abouttwelve (12) months, about twenty-four (24) months, or longer. In someembodiments, the period of time is within the range of about one (1) dayto about twenty-four (24) months, about two (2) weeks to about twelve(12) months, about two (2) months to about five (5) months, etc. Forexample, if a nanoparticle composition is subjected to prolongedstorage, temperature changes, and/or pH changes and a majority of thenanoparticles in the population maintain a diameter within a statedrange (i.e., for example, between approximately 10 nm-120 nm), thenanoparticle composition is stable. For some such populations, amajority is more than about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%,about 99.6%, about 99.7%, about 99.8%, about 99.9%, or more. In someembodiments of the invention, where a nanoparticle composition comprisesone or more biologically active agents (e.g. botulinum toxin), thenanoparticle composition is considered stable if the concentration ofbiologically active agent is maintained in the composition over thedesignated period of time under a designated set of conditions.

Substantially free of An inventive nanoparticle composition is said tobe “substantially free of” particles whose diameter is outside of astated range when no more than about 50% of the particles in thatcomposition have diameters outside of the range. In some embodiments, nomore than 25% of the particles are outside of the range. In someembodiments, no more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the particleshave diameters outside of the stated range.

Suffering from: An individual who is “suffering from” a disease,disorder, or condition (e.g., facial wrinkles) has been diagnosed withor exhibits symptoms of the disease, disorder, or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of inventive AEnanoparticle composition that is sufficient, when administered to apatient suffering from or susceptible to a disease, disorder, and/orcondition, to treat the disease, disorder, and/or condition.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect.

Toxic solvent: As used herein, the term “toxic solvent” refers to anysubstance that may alter, disrupt, remove, or destroy an animal'stissue. As would be understood by one of ordinary skill in the art, ananimal's tissue can include living cells, dead cells, extracellularmatrix, cellular junctions, biological molecules, etc. To give but a fewexamples, toxic solvents include dimethyl sulfoxide, dimethyl acetimide,dimethyl foramide, chloroform, tetramethyl foramide, acetone, acetates,and alkanes.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a biologically active agentthat partially or completely alleviates, ameliorates, relives, inhibits,delays onset of, reduces severity of, and/or reduces incidence of one ormore symptoms or features of a particular disease, disorder, and/orcondition (e.g., facial wrinkles). Such treatment may be of a subjectwho does not exhibit signs of the relevant disease, disorder and/orcondition and/or of a subject who exhibits only early signs of thedisease, disorder, and/or condition. Alternatively or additionally, suchtreatment may be of a subject who exhibits one or more established signsof the relevant disease, disorder and/or condition.

Uniform: The term “uniform,” when used herein in reference to ananoparticle composition, refers to a nanoparticle composition in whichthe individual nanoparticles have a specified range of particle diametersizes. For example, in some embodiments, a uniform nanoparticlecomposition is one in which the difference between the minimum diameterand maximum diameter does not exceed approximately 600, approximately550, approximately 500, approximately 450, approximately 400,approximately 350, approximately 300, approximately 250, approximately200, approximately 150, approximately 100, approximately 90,approximately 80, approximately 70, approximately 60, approximately 50,or fewer nm. In some embodiments, particles (e.g., AE nanoparticles)within inventive uniform nanoparticle compositions have diameters thatare smaller than about 600, about 550, about 500, about 450, about 400,about 350, about 300, about 250, about 200, about 150, about 130, about120, about 115, about 110, about 100, about 90, about 80 nm, or less. Insome embodiments, particles (e.g., AE nanoparticles) within inventiveuniform nanoparticle compositions have diameters within the range ofabout 10 and about 600 nanometers. In some embodiments, particles (e.g.,AE nanoparticles) within inventive uniform nanoparticle compositionshave diameters within the range of about 10 to about 300, about 10 toabout 200, about 10 to about 150, about 10 to about 130, about 10 toabout 120, about 10 to about 115, about 10 to about 110, about 10 toabout 100, or about 10 to about 90 nm. In some embodiments, particles(e.g., AE nanoparticles) within inventive botulinum nanoparticlecompositions have an average particle size that is under about 300,about 250, about 200, about 150, about 130, about 120, about 115, about110, about 100, or about 90 nm. In some embodiments, the averageparticle size is within the range of about about 10 to about 300, about50 to about 250, about 60 to about 200, about 65 to about 150, about 70to about 130 nm. In some embodiments, the average particle size is about80 to about 110 nm. In some embodiments, the average particle size isabout 90 to about 100 nm. In some embodiments, a majority of theparticles (e.g., AE nanoparticles) within inventive uniform nanoparticlecompositions have diameters below a specified size or within a specifiedrange. In some embodiments, the majority is more than 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more of the particles in the composition. In some embodimentsof the invention, a uniform nanoparticle composition is achieved bymicrofluidization of a sample. In some embodiments of the invention, auniform nanoparticle composition is prepared by exposure to high shearforce, e.g., by microfluidization.

Vector: As used herein, “vector” refers to a nucleic acid molecule whichcan transport another nucleic acid to which it has been linked. In someembodiment, vectors can achieve extra-chromosomal replication and/orexpression of nucleic acids to which they are linked in a host cell suchas a eukaryotic and/or prokaryotic cell. Vectors capable of directingthe expression of operatively linked genes are referred to herein as“expression vectors.”

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS Amphiphilic Entity (AE)Nanoparticles

In some embodiments, the present invention provides compositionscontaining AE nanoparticles. In some embodiments, the nanoparticlecompositions are stable (i.e. the particles of the nanoparticlecomposition stay within a stated range over time and when subjected totemperature and/or pH changes). In some embodiments, the nanoparticlecompositions are sterile (i.e. the nanoparticle composition contains noliving cellular contaminants). In some embodiments, the nanoparticlecompositions are bacteria-resistant (i.e. the nanoparticle compositionsare characterized by no observable bacterial growth). In someembodiments, the nanoparticle compositions comprising AE nanoparticlesare completely free or substantially free of toxic components. In someembodiments, the AE nanoparticles are completely free or substantiallyfree of toxic components.

In some embodiments, the nanoparticle compositions are uniform. In someembodiments, a uniform nanoparticle composition comprises a populationof particles whose difference between the minimum and maximum diametersdoes not exceed approximately 600 nm, approximately 550 nm,approximately 500 nm, approximately 450 nm, approximately 400 nm,approximately 350 nm, approximately 300 nm, approximately 250 nm,approximately 200 nm, approximately 150 nm, or approximately 100 nm.

In some embodiments, AE nanoparticles inventive nanoparticlecompositions have diameters that are smaller than about 1000, about 600,about 550, about 500, about 450, about 400, about 350, about 300, about250, about 200, about 150, about 130, about 120, about 115, about 110,about 100, about 90, about 80, about 50 nm, or less.

In some embodiments, inventive AE nanoparticles have a diameter of 1 to1000 nm, 1 to 600 nm, 1 to 500 nm, 1 to 400 nm, 1 to 300 nm, 1 to 200nm, 1 to 150 nm, 1 to 120 nm, 1 to 100 nm, 1 to 75 nm, 1 to 50 nm, or 1to 25 nm. In some embodiments, inventive AE nanoparticles have adiameter of 1 to 15 nm, 15 to 200 nm, 25 to 200 nm, 50 to 200 nm, or 75to 200 nm.

In some embodiments, the total particle distribution is encompassedwithin the specified range of particle diameter size. In someembodiments, less than 50%, 25%, 10%, 5%, or 1% of the total particledistribution is outside of the specified range of particle diametersizes. In some embodiments, less than 1% of the total particledistribution is outside of the specified range of particle diametersizes. In certain embodiments, the nanoparticle composition issubstantially free of particles having a diameter larger than 300 nm,250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm, 50 nm, or 25 nm.

In some embodiments, AE nanoparticles within inventive nanoparticlecompositions have an average particle size that is under about 300,about 250, about 200, about 150, about 130, about 120, about 115, about110, about 100, about 90, or about 50 nm. In some embodiments, theaverage particle size is within the range of about 10 to about 300,about 50 to about 250, about 60 to about 200, about 65 to about 150,about 70 to about 130 nm. In some embodiments, the average particle sizeis about 80 to about 110 nm. In some embodiments, the average particlesize is about 90 to about 100 nm.

In some embodiments, inventive nanoparticle compositions aresubstantially free of particles having a diameter in excess of 300 nm.Specifically, in some embodiments, fewer than 50%, of the nanoparticlesin inventive nanoparticle compositions have a diameter in excess of 300nm. In some embodiments, fewer than 25% of the particles have a diameterin excess of 300 nm. In some embodiments, fewer than 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or less of the particles have a diameter in excess of 300 nm.Furthermore, in some embodiments, the nanoparticles in inventivenanoparticle compositions have diameters within the range of 10 to 300nm.

In some embodiments, inventive nanoparticle compositions aresubstantially free of particles having a diameter in excess of 200 nm.Specifically, in some embodiments, fewer than 50%, of the nanoparticlesin inventive nanoparticle compositions have a diameter in excess of 200nm. In some embodiments, fewer than 25% of the particles have a diameterin excess of 200 nm. In some embodiments, fewer than 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or less of the particles have a diameter in excess of 200 nm.Furthermore, in some embodiments, the nanoparticles in inventivenanoparticle compositions have diameters within the range of 10 to 200nm.

In some embodiments, inventive nanoparticle compositions aresubstantially free of particles having a diameter in excess of 120 nm.Specifically, in some embodiments, fewer than 50%, of the nanoparticlesin inventive nanoparticle compositions have a diameter in excess of 120nm. In some embodiments, fewer than 25% of the particles have a diameterin excess of 120 nm. In some embodiments, fewer than 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or less of the particles have a diameter in excess of 120 nm.Furthermore, in some embodiments, the nanoparticles in inventivenanoparticle compositions have diameters within the range of 10 to 120nm.

In some embodiments, a majority of the AE nanoparticles within inventivecompositions have diameters below a specified size or within a specifiedrange. In some embodiments, the majority is more than 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more of the particles in the composition.

Zeta potential is a measurement of the electric potential at a shearplane. A shear plane is an imaginary surface separating a thin layer ofliquid bound to a solid surface (e.g. the surface of inventivenanoparticles) and showing elastic behavior from the rest of liquid(e.g. liquid dispersion medium) showing normal viscous behavior. In someembodiments, inventive AE nanoparticles have a zeta potential rangingbetween −50 mV to +50 mV. In some embodiments, inventive AEnanoparticles have a zeta potential ranging between −25 mV to +25 mV. Insome embodiments, inventive AE nanoparticles have a zeta potentialranging between −10 mV to +10 mV.

In some embodiments, the AE nanoparticles are micelles. In someembodiments, the AE nanoparticles provided in accordance with thepresent invention are nanospheres. In some embodiments, the AEnanoparticles comprise one or more amphiphilic entities. Inventive AEnanoparticles may optionally comprise one or more dispersion media,surfactants, biologically active agents, or release-retardingingredients.

Inventive nanoparticle compositions may be emulsions or dispersions. Insome embodiments, the compositions are “oil-in-water” dispersions (i.e.,dispersions in which oily particles are dispersed within an aqueousdispersion medium); in some embodiments, the compositions are“water-in-oil” dispersions (i.e., dispersions in which aqueous particlesare dispersed within an oily dispersion medium). In some embodiments,some or all of the nanoparticles have a micellar structure in which alumen is enclosed by a micellar “membrane.” In some such embodiments,the lumen of the micelle has the same character (e.g., aqueous vs. oily)as the dispersion medium and the micellar membrane has the opposingcharacter (e.g., oily vs. aqueous); in some embodiments, the lumen ofthe micelle has the same character as the micellar membrane and thedispersion medium has the opposing character.

Premix

In some embodiments, the invention provides methods of preparing theinventive AE nanoparticles. The method generally involves combining oneor more amphiphilic entities to form a “premix” and applying high shearforces to the premix. The premix generally contains one or moreamphiphilic entities and one or more dispersion media. In someembodiments, the premix may also contain one or more additionalsubstances such as, for example, surfactants, biologically activeagents, release-retarding ingredients, etc. Those of ordinary skill inthe art, however, will appreciate that it is not essential that allcomponents necessarily be present in the premix (or be present in theirtotal eventual amount) in the premix; in some instances it may bedesirable or appropriate to add or to supplement one or more componentslater.

In some embodiments, the premix components are selected and/ormaintained under conditions that permit nanoparticle formation beforethe high shear forces are applied. In some embodiments, the premix isnot maintained under conditions that permit particle formation beforethe high shear forces are applied. In some embodiments, these particlesare nano- or micro-particles. In some embodiments, these particles aremicelles. In some embodiments, nanoparticles are allowed to form fromthe premix components before the high shear forces are applied. In someembodiments, nanoparticle formation in the premix is inhibited beforethe high shear forces are applied. In certain embodiments, the highshear forces are applied after nanoparticles have formed from the premixcomponents. In certain embodiments, the high shear forces are appliedbefore nanoparticles form from the premix components. In certainembodiments, the high shear forces are applied while nanoparticles areforming from the premix components.

In some embodiments, the present invention provides methods ofmanufacturing AE nanoparticles. The method generally involves preparinga premix of two or more amphiphilic entities and subjecting the premixto high shear forces. In some embodiments, the premix may comprise oneor more dispersion media, surfactants, biologically active agents, orrelease-retarding ingredients.

In some embodiments, the methods of producing inventive AE nanoparticlesinvolve steps of providing a premix, allowing or inducing assembly ofparticles (e.g., nanoparticles, microparticles, and/or micelles) withinthe premix, and subjecting the particles to high shear forces such thatan inventive nanoparticle composition is obtained. In some embodiments,particle formation in the premix may be by emulsion polymerization,self-assembly, or by any other known technique for producing micro- ornano-particles.

In certain embodiments, particle formation in the premix may involve thesteps of dissolving an amphiphilic entity in a dispersion medium,gradually adding water to the solution of dispersion medium andamphiphilic entity, and waiting for particles (e.g. nanoparticles,microparticles, and/or micelles) to self-assemble. In some embodiments,particle formation can be induced by slowly cooling the solution.

In certain embodiments, particle formation in the premix may involve thesteps of dissolving an amphiphilic entity in a dispersion medium such aswater, stirring for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 hours whileallowing particles (e.g. nanoparticles, microparticles, and/or micelles)to form, dialyzing against water to remove any organic dispersion mediumto stabilize the particles, and freeze-drying to produce a composition.

In some embodiments of the present invention that utilize a premix, itis to be understood that the premix components may assemble intoparticles before the application of high shear force. At least some ofsuch particles may be microparticles or even nanoparticles. In someembodiments, an inventive nanoparticle composition is prepared from apremix, wherein the premix is selected from the group comprising asuspension or a microemulsion. In some embodiments, however, particlestructures do not form in the premix before application of high shearforce.

In some embodiments of the present invention, all of the componentspresent in the final nanoparticle composition are present in the premixand are subjected to high shear force to produce the nanoparticlecomposition. In some embodiments of the present invention, one or moreof the components that are present in the final nanoparticle compositionis/are missing from the premix or is/are present in the premix in asmaller amount than in the final nanoparticle composition. That is, insome embodiments of the present invention, one or more materials areadded to the nanoparticle composition after the premix is subjected tohigh shear stress.

In certain embodiments of the invention, the premix is prepared as asolution prior to application of high shear force. In particular, fornanoparticle compositions that include at least one biologically activeagent (e.g., an unmodified peptide), it is often desirable for thebiologically active agent to be dissolved in the premix before the highshear stress is applied. Thus, in many embodiments, the biologicallyactive agent is soluble in at least one of the media (or in acombination of media utilized in the premix). In some embodiments of theinvention, such dissolution requires heating; in other embodiments itdoes not.

Below, we discuss exemplary components of a premix that is subjected tohigh shear force according to the present invention.

Amphiphilic Entities

The present invention provides nanoparticles and nanoparticlecompositions comprising amphiphilic entities. Useful amphiphilicentities include natural entities, synthetic entities, and entities thatcontain both natural and synthetic components. In some embodiments,amphiphilic entities may comprise one or more polymers, and/or one ormore compounds with polymeric character.

As discussed above, an amphiphilic entity is one that has bothhydrophobic and hydrophilic natures. As will be appreciated by those ofordinary skill in the art, an amphiphilic entity can be comprised in anynumber of different ways. In some embodiments, an amphiphilic entity maycomprise one or more individual compounds or substances that is itselfamphiphilic. To give but a few examples, such compounds or moleculesinclude polyethylene glycol (PEG), phospholipids, cholesterols,glycolipids fatty acids, bile acids, and saponins. PEG is generallyrecognized as safe for use in food, cosmetics, and medicines by the USFood and Drug Administration. PEG is water-soluble, non-toxic, odorless,lubricating, nonvolatile, and nonirritating.

In some embodiments, an amphiphilic entity may comprise one or moreindividual components that is not itself amphiphilic but that has somehydrophilic or hydrophobic character. In such embodiments, two or moresuch non-amphiphilic components will typically be associated with oneanother such that the assemblage of the individual components isamphiphilic. Such association may or may not involve covalent linkage;such association may involve non-covalent bonding (e.g., viaelectrostatic interactions, affinity interactions, hydrophobicinteractions, hydrogen bonding, Van der Waals interactions, ionicinteraction, dipole-dipole interaction, etc.). In general, suchassociation may involve any relevant force, bond, or means of adhesion.

In some embodiments, an amphiphilic entity of the present invention maybe constructed from two or more individual components having differingdegrees of hydrophilicity or hydrophobicity. In certain embodiments, anamphiphilic entity may comprise at least one hydrophilic component andat least one hydrophobic component. In certain embodiments, the“hydrophilic” and “hydrophobic” components are either hydrophilic orhydrophobic relative to one another.

In some embodiments, two or more components of differing degrees ofhydrophilicity or hydrophobicity may be bonded together by covalentbonds to form a homopolymer or a co-polymer. In some embodiments, aco-polymer may be a block co-polymer. In some embodiments, a co-polymermay be a graft co-polymer.

In some embodiments, an amphiphilic entity may comprise or consist of anamphiphilic block co-polymer. In some embodiments, an amphiphilic blockco-polymer may be a diblock co-polymer. In certain embodiments, anamphiphilic diblock co-polymer may comprise a first polymer block and asecond polymer block connected covalently at the chain ends. In specificembodiments, the first polymer block may comprise repeating units of ahydrophilic component, and the second polymer block may compriserepeating units of a hydrophobic component. In specific embodiments, thefirst polymer block may comprise repeating units of a hydrophobiccomponent, and the second polymer block may comprise repeating units ofa hydrophilic component. In some embodiments, an amphiphilic blockco-polymer may be a multiblock co-polymer. In certain embodiments, anamphiphilic block co-polymer may comprise multiple alternating blocks oftwo or more polymers connected covalently at the chain ends. In specificembodiments, an amphiphilic block co-polymer may comprise multiplealternating hydrophilic blocks and hydrophobic blocks connectedcovalently at the chain ends. In specific embodiments, each block of thealternating blocks may comprise repeating units of either hydrophiliccomponents or hydrophobic components.

In some embodiments, an amphiphilic entity may comprise or consist of anamphiphilic graft co-polymer. In some embodiments, an amphiphilic graftco-polymer may comprise or consist of blocks of polymers connectedcovalently to the side chains of other blocks of polymers. In specificembodiments, each polymer block may comprise or consist of repeatingunits of either hydrophilic or hydrophobic components. In certainembodiments, an amphiphilic graft co-polymer may comprise or consist ofa first polymer block and a second polymer block connected covalently toa side chain of the first polymer block. In certain embodiments, thefirst polymer block may comprise or consist of repeating units of ahydrophilic component, and the second block may comprise repeating unitsof a hydrophobic component. In certain embodiments, the first polymerblock may comprise or consist of repeating units of a hydrophobiccomponent, and the second block may comprise repeating units of ahydrophilic component.

In some embodiments, an amphiphilic block or graft co-polymer mayinclude a hydrophilic polymer block comprising repeating units of apolysaccharide and a hydrophobic polymer block comprising repeatingunits of a polyester or polysaccharide. Alternatively or additionally,an amphiphilic block or graft co-polymer may include a hydrophobicpolymer block comprising repeating units of a polysaccharide and ahydrophilic polymer block comprising repeating units of a polyester orpolysaccharide. Such a hydrophilic polymer block can contain repeatingunits of any type of hydrophilic polymer, such as a polysaccharide (e.g.pullulan) or polyalkene oxide (e.g. polyethylene oxide). The hydrophobicpolymer block can contain repeating units of any type of hydrophobicpolymer, such as a polycaprolactone, poly (lactic acid), poly (glycolicacid), poly dioxanone, copolymers of these or polyamide (e.g.polycaprolactam).

In some embodiments, the hydrophilic portion of the amphiphilic entitymay be non-ionic. In some embodiments, the hydrophilic component of anamphiphilic entity comprises one or more ionic groups. In general, suchionic groups are hydrophilic and can confer hydrophilic nature on theamphiphilic entity.

In some embodiments, the ionic group may be cationic. In someembodiments, the cationic group may be an ammonium (NH₄ ⁺), nitronium(NO₂ ⁺), nitrosyl (NO⁺), hydronium (H₃O⁺), mercurous (Hg₂ ²⁺),phosphonium (PH₄ ⁺), vanadyl (VO²⁺), or salt thereof.

In some embodiments, the ionic group may be anionic. In someembodiments, the anionic group may be a fatty acid, arsenide (As³⁻),azide (N₃ ⁻), bromide (Br⁻), chloride (Cl⁻), fluoride (F⁻), hydride(H⁻), iodide (I⁻), nitride (N³⁻), oxide (O²⁻), phosphide (P³⁻), selenide(Se²⁻), sulfide (S²⁻), peroxide (O₂ ²⁻), arsenate (AsO₄ ³⁻), arsenite(AsO₃ ³⁻), borate (BO₃ ³⁻), perbromate (BrO₄ ⁻), bromate (BrO₃ ⁻),bromite (BrO₂ ⁻), hypobromite (BrO⁻), carbonate (CO₃ ²⁻), hydrogencarbonate (HCO₃ ⁻), chlorate (ClO₃ ⁻), perchlorate (ClO₄ ⁻), chlorite(ClO₂ ⁻), hypochlorite (ClO⁻), chromate (CrO₄ ²⁻), dichromate (Cr₂O₇²⁻), perfluorate (BrO₄ ⁻), fluorate (BrO₃ ⁻), fluorite (BrO₂ ⁻),hypofluorite (BrO⁻), periodate (IO₄ ⁻), iodate (IO₃ ⁻), iodite (IO₂ ⁻),hypoiodite (IO⁻), nitrate (NO₃ ⁻), nitrite (NO₂ ⁻), phosphate (PO₄ ³⁻),hydrogen phosphate (HPO₄ ²⁻), dihydrogen phosphate (H₂PO₄ ⁻), phosphite(PO₃ ³⁻), silicate (SiO₃ ²⁻), sulfate (SO₄ ²⁻), thiosulfate (S₂O₃ ²⁻),hydrogen sulfate (HSO₄ ⁻), sulfite (SO₃ ²⁻), hydrogen sulfite (HSO₃ ⁻),sulfonate (—S(═O)₂—O⁻), acetate (C₂H₃O₂ ⁻), formate (HCO₂ ⁻), oxalate(C₂O₄ ²⁻), hydrogen oxalate (HC₂O₄ ⁻), citrate (C₆H₅O₇ ³⁻), succinate(C₄H₄O₄ ²⁻), fumarate (C₄H₂O₄ ²⁻), malate (C₄H₅O₅ ²⁻), hydrogen sulfide(HS⁻), telluride (Te²⁻), amide (NH₂ ⁻), cyanate (OCN⁻), thiocyanate(SCN⁻), cyanide (CN⁻), hydroxide (OH⁻), permanganate (MnO₄ ⁻), or saltthereof.

In some embodiments, the hydrophilic component of an amphiphilic entitymay comprise or consist of a nucleic acid. For example, the nucleic acidpolymer may include DNA, RNA, or combinations thereof. In someembodiments, the nucleic acid polymer may be an oligonucleotide and/orpolynucleotide. In some embodiments, the nucleic acid polymer may be anoligonucleotide and/or modified oligonucleotide; an antisenseoligonucleotide and/or modified antisense oligonucleotide; a cDNA; agenomic DNA; viral DNA and/or RNA; DNA and/or RNA chimeras; plasmids;cosmids; gene fragments; an artificial and/or natural chromosome (e.g. ayeast artificial chromosome) and/or a part thereof; an RNA (e.g. anmRNA, a tRNA, an rRNA and/or a ribozyme); a peptide nucleic acid (PNA);a polynucleotide comprising synthetic analogues of nucleic acids, whichmay be modified or unmodified; various structural forms of DNA includingsingle-stranded DNA, double-stranded DNA, supercoiled DNA and/ortriple-helical DNA; Z-DNA; and/or combinations thereof.

In some embodiments, the hydrophilic component of an amphiphilic entitymay comprise or consist of a carbohydrate. In some embodiments, thecarbohydrate may be a polysaccharide composed of simple sugars (or theirderivatives) connected by glycosidic bonds, as known in the art. Suchsugars may include, but are not limited to, glucose, fructose,galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose,mannose, xylose, arabinose, glucoronic acid, galactoronic acid,mannuronic acid, glucosamine, galatosamine, and neuramic acid. In someembodiments, the polymer may be a hydrophilic carbohydrate, includingaminated, carboxylated, and sulfated polysaccharides. In someembodiments, the hydrophilic carbohydrate may be one or more ofpullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxycellulose, methylcellulose, dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,starch, chitin, heparin, konjac, glucommannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan. In some embodiments, hydrophilicpolysaccharides can be modified to become hydrophobic by introducing alarge number of side-chain hydrophobic groups. In some embodiments, ahydrophobic carbohydrate may include cellulose acetate, pullulanacetate, konjac acetate, amylose acetate, and dextran acetate.

In some embodiments, the hydrophilic component of an amphiphilic entitymay comprise or consist of a gum including, but not limited to, xanthangum, alginic acid, caraya gum, sodium alginate, and/or locust bean gum.

In some embodiments, a component of an amphiphilic entity may compriseor consist of a protein. In some embodiments, a protein is a hydrophiliccomponent of an amphiphilic entity. In other embodiments, a protein is ahydrophobic component of an amphiphilic entity. Exemplary proteins thatmay be used in accordance with the present invention include, but arenot limited to, albumin, collagen, or a poly(amino acid) (e.g.polylysine).

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of one or more fatty acid groups or saltsthereof. In general, such groups are typically hydrophobic and canconfer hydrophobic nature onto the amphiphilic entity. In someembodiments, the fatty acid group may comprise digestible, long chain(e.g., C₈-C₅₀), substituted or unsubstituted hydrocarbons. In someembodiments, the fatty acid group may be a C₁₀-C₂₀ fatty acid or saltthereof. In some embodiments, the fatty acid group may be a C₁₅-C₂₀fatty acid or salt thereof. In some embodiments, the fatty acid groupmay be a C₁₅- C₂₅ fatty acid or salt thereof. In some embodiments, thefatty acid group may be unsaturated. In some embodiments, the fatty acidgroup may be monounsaturated. In some embodiments, the fatty acid groupmay be polyunsaturated. In some embodiments, a double bond of anunsaturated fatty acid group may be in the cis conformation. In someembodiments, a double bond of an unsaturated fatty acid may be in thetrans conformation.

In some embodiments, the fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, the fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of one or more biocompatible and/orbiodegradable synthetic polymers, including, for example, polyethylenes,polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.poly(sebacic anhydride)), polyhydroxyacids (e.g.poly(β-hydroxyalkanoate)), polypropylfumerates, polycaprolactones,polyamides (e.g. polycaprolactam), polyacetals, polyethers, polyesters(e.g. polylactide and polyglycolide), biodegradable polycyanoacrylates,polyvinyl alcohols, and biodegradable polyurethanes. For example, theamphiphilic entity may comprise one or more of the followingbiodegradable polymers: poly(lactic acid), poly(glycolic acid),poly(caprolactone), poly(lactide-co-glycolide),poly(lactide-co-caprolactone), poly(glycolide-co-caprolactone), andpoly(DL-lactide-co-glycolide).

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of one or more acrylic polymers. In certainembodiments, acrylic polymers include, for example, acrylic acid andmethacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethylmethacrylates, cyanoethyl methacrylate, aminoalkyl methacrylatecopolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acidalkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acidanhydride), methyl methacrylate, polymethacrylate, poly(methylmethacrylate) copolymer, polyacrylamide, aminoalkyl methacrylatecopolymer, glycidyl methacrylate copolymers, and combinations comprisingone or more of the foregoing polymers. The acrylic polymer may comprisefully-polymerized copolymers of acrylic and methacrylic acid esters witha low content of quaternary ammonium groups.

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of a polyester. Exemplary such polyestersinclude, for example, polyalkylene glycols, poly(glycolide-co-lactide),PEGylated poly(lactic-co-glycolic acid), poly(lactic acid), PEGylatedpoly(lactic acid), poly(glycolic acid), PEGylated poly(glycolic acid),co-polymers of polylactic and polyglycolic acid, and derivativesthereof. In some embodiments, polyesters include, for example,polyanhydrides, poly(ortho ester) PEGylated poly(ortho ester),poly(caprolactone), PEGylated poly(caprolactone), polylysine, PEGylatedpolylysine, poly(ethylene imine), PEGylated poly(ethylene imine), andderivatives thereof. In some embodiments, polyesters may include, forexample, polycaprolactone, poly(L-lactide-co-L-lysine), poly(serineester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, amphiphilic entities may have biological activity.

Those skilled in the art will recognize that this is an exemplary, notcomprehensive, list of amphiphilic entities. The percent of amphiphilicentity in the composition from which AE nanoparticles are prepared(e.g., in the premix) can range from 40% to 99%, from 50% to 99%, from60% to 99%, from 70% to 99%, from 80% to 99%, from 80% to 90%, or from90% to 99%. In some embodiments the percent of amphiphilic entity in thecomposition from which AE nanoparticles are prepared (e.g., in thepremix) is approximately 75%, approximately 76%, approximately 77%,approximately 78%, approximately 79%, approximately 80%, approximately81%, approximately 82%, approximately 83%, approximately 84%,approximately 85%, approximately 86%, approximately 87%, approximately88%, approximately 89%, approximately 90%, approximately 91%,approximately 92%, approximately 93%, approximately 94%, approximately95%, approximately 96%, approximately 97%, approximately 98%, orapproximately 99%.

Dispersion Media

In general, the premix is expected to contain at least one dispersionmedium. In some embodiments, the premix may contain a hydrophilicdispersion medium. In some embodiments, the premix may contain ahydrophobic dispersion medium. In some embodiments, the premix maycontain a combination of two or more dispersion media, for example ofdifferent characters. In some embodiments, the premix may contain atleast two immiscible dispersion media.

It will be appreciated that the selection of appropriate dispersionmedium will depend, at least in part, on the nature of the amphiphilicentit(ies) being employed and on whether it is intended for the intendedexternal and internal character of the intended nanoparticles. Forexample, in some embodiments, the nanoparticles will have hydrophilicexternal character and hydrophobic internal character; in someembodiments, the nanoparticles will have hydrophobic external characterand hydrophilic character; in some embodiments, the nanoparticles willhave hydrophilic external and internal character; and in someembodiments, the nanoparticles with have hydrophobic external andinternal character. It will be appreciated by those of ordinary skill inthe art that the same nanoparticle components can sometimes assembleinto different nanoparticle structures, having different internal andexternal characters. To give but one example, nanoparticles formed froma monolayer of an individual amphiphilic compound will have differingexternal and internal characters, whereas nanoparticles formed from abilayer of the same compound will have the same external and internalcharacter.

In some embodiments of the invention, hydrophilic dispersion media areutilized. In some embodiments, such hydrophilic dispersion media areaqueous. Such aqueous dispersion media include but are not limited to,water, short chain alcohols (e.g. ethanol), oils, 5% dextrose, Ringer'ssolutions (e.g. lactated Ringer's injection, lactated Ringer's plus 5%dextrose injection, aceylated Ringer's injection), Normosol-M, IsolyteE, dimethyl sulfoxide (DMSO), dimethyl acetamide, dimethyl formamide,chloroform, tetramethyl formamide, carbon tetrachloride, N-methylpyrolidone, or dichloroethane, and the like, and combinations thereof.

In some embodiments of the invention, hydrophobic dispersion media areutilized. In some embodiments, such hydrophobic dispersion media areoils. In general, any oil known in the art is suitable for use in makingthe inventive AE nanoparticles. In some embodiments, the oil maycomprise one or more fatty acid groups or salts thereof. In someembodiments, the fatty acid group may comprise digestible, long chain(e.g., C₈-C₅₀), substituted or unsubstituted hydrocarbons. In someembodiments, the fatty acid group may be a C₁₀-C₂₀ fatty acid or saltthereof. In some embodiments, the fatty acid group may be a C₁₅-C₂₀fatty acid or salt thereof. In some embodiments, the fatty acid groupmay be a C₁₅-C₂₅ fatty acid or salt thereof. In some embodiments, thefatty acid group may be unsaturated. In some embodiments, the fatty acidgroup may be monounsaturated. In some embodiments, the fatty acid groupmay be polyunsaturated. In some embodiments, a double bond of anunsaturated fatty acid group may be in the cis conformation. In someembodiments, a double bond of an unsaturated fatty acid may be in thetrans conformation.

In some embodiments, the fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, the fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, the oil is a liquid triglyceride. In certainembodiments, the oil is a medium chain (e.g., 6-12 carbons) triglyceride(e.g., Labrafac WL 1349, coconut oil, palm kernel oil, camphor treedrupe oil, etc.). In certain embodiments, the oil is a short chain(e.g.,2-5 carbons) triglyceride. In certain embodiments, the oil is a longchain (e.g., greater than 12 carbons) triglyceride (e.g., soybean oil,sunflower oil, etc.).

Suitable oils for use with the present invention include, but are notlimited to, almond, apricot kernel, avocado, babassu, bergamot, blackcurrent seed, borage, cade, camomile, canola, caraway, carnauba, castor,cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed,emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,grape seed, hazel nut, hyssop, jojoba, kukui nut, lavandin, lavender,lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoamseed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel,peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran,rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn,sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle,tsubaki, vetiver, walnut, and wheat germ oils, and combinations thereof.Suitable oils for use with the present invention include, but are notlimited to, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, andcombinations thereof.

Those of ordinary skill in the art will appreciate that the term“dispersion medium” is not intended to imply a particular amount of thematerial be present. For example, particularly in a system that utilizestwo or more dispersion media (e.g., with differenthydrophobic/hydrophilic character), the relative amounts of differentdispersion media may be adjusted as desired. For example, the percent ofdispersion medium in the composition from which AE nanoparticles areprepared (e.g., in the premix) can range from 0% to 99%, from 10% to99%, from 25% to 99%, from 50% to 99%, or from 75% to 99%. In someembodiments, the percent of dispersion medium in the composition fromwhich AE nanoparticles are prepared (e.g., in the premix) can range from0% to 75%, from 0% to 50%, from 0% to 25%, or from 0% to 10%.

In some embodiments, the percent of oil in the composition from which AEnanoparticles are prepared (e.g., in the premix) ranges between 0% to30%. In some embodiments the percent of oil in the composition fromwhich AE nanoparticles are prepared (e.g., in the premix) isapproximately 1%, approximately 2%, approximately 3%, approximately 4%,approximately 5%, approximately 6%, approximately 7%, approximately 9%,approximately 10%, approximately 11%, approximately 12%, approximately13%, approximately 14%, approximately 15%, approximately 16%,approximately 17%, approximately 18%, approximately 19%, approximately20%, approximately 21%, approximately 22%, approximately 23%,approximately 24%, approximately 25%, approximately 26%, approximately27%, approximately 28%, approximately 29%, or approximately 30%. In someembodiments the percent of oil is approximately 8%. In some embodimentsthe percent of oil is approximately 5%.

In some embodiments, the premix comprises oil and surfactant at a ratioranging between 0.5:1 to 10:1. In some embodiments, the ratio of oil tosurfactant is approximately 0.5:1, approximately 1:1, approximately 2:1,approximately 3:1, approximately 4:1, approximately 5:1, approximately6:1, approximately 7:1, approximately 8:1, approximately 9:1, orapproximately 10:1. In some embodiments, the ratio of surfactant to oilis approximately 0.5:1, approximately 1:1, approximately 2:1,approximately 3:1, approximately 4:1, approximately 5:1, approximately6:1, approximately 7:1, approximately 8:1, approximately 9:1, orapproximately 10:1. In some embodiments, the premix comprises oil andsurfactant at a ratio ranging between 0.5:1 to 2:1. In some embodiments,the ratio of oil to surfactant is approximately 0.5:1, approximately1:1, or approximately 2:1. In some embodiments, the ratio of surfactantto oil is approximately 0.5:1, approximately 1:1, or approximately 2:1.In certain embodiments, the ratio of oil to surfactant is approximately1:1.

Those skilled in the art will recognize that the above presents certainexemplary, not comprehensive, lists of possible dispersion media for usein accordance with the present invention. Any appropriate dispersionmedium may be used in the production of AE nanoparticles.

Surfactants

In some embodiments, the premix may optionally comprise one or moresubstances with surfactant activity. In some embodiments, a substancewith surfactant activity can promote the production of AE nanoparticleswith increased stability, improved uniformity, or increased viscosity.Surfactants can be particularly useful in embodiments that utilize twoor more dispersion media. The percent of substances with surfactantactivity in the composition from which AE nanoparticles are prepared(e.g., in the premix) can range from 0% to 99%, from 10% to 99%, from25% to 99%, from 50% to 99%, or from 75% to 99%. In some embodiments,the percent of substances with surfactant activity in the compositionfrom which AE nanoparticles are prepared (e.g., in the premix) can rangefrom 0% to 75%, from 0% to 50%, from 0% to 25%, or from 0% to 10%.

In some embodiments, the percent of substances with surfactant activityin the composition from which AE nanoparticles are prepared (e.g., inthe premix) ranges between 0% to 30%. In some embodiments the percent ofsubstances with surfactant activity in the composition from which AEnanoparticles are prepared (e.g., in the premix) is approximately 1%,approximately 2%, approximately 3%, approximately 4%, approximately 5%,approximately 6%, approximately 7%, approximately 9%, approximately 10%,approximately 11%, approximately 12%, approximately 13%, approximately14%, approximately 15%, approximately 16%, approximately 17%,approximately 18%, approximately 19%, approximately 20%, approximately21%, approximately 22%, approximately 23%, approximately 24%,approximately 25%, approximately 26%, approximately 27%, approximately28%, approximately 29%, or approximately 30%. In some embodiments thepercent of substances with surfactant activity is approximately 8%. Insome embodiments the percent of substances with surfactant activity isapproximately 5%.

Any substance with surfactant activity known in the art is suitable foruse in making the inventive AE nanoparticles. Such surfactants include,but are not limited to, phosphoglycerides; phosphatidylcholines;dipalmitoyl phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine(DOPE); dioleyloxypropyltriethylammonium (DOTMA);dioleoylphosphatidylcholine; cholesterol; cholesterol ester;diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG);hexanedecanol; fatty alcohols such as polyethylene glycol (PEG);polyoxyethylene-9-lauryl ether; a surface active fatty acid, such aspalmitic acid or oleic acid; fatty acids; fatty acid monoglycerides;fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span 85)glycocholate; sorbitan monolaurate (Span 20); polysorbate 20 (Tween-20);polysorbate 60 (Tween-60); polysorbate 65 (Tween-65); polysorbate 80(Tween-80); polysorbate 85 (Tween-85); polyoxyethylene monostearate;surfactin; a poloxomer; a sorbitan fatty acid ester such as sorbitantrioleate; lecithin; lysolecithin; phosphatidylserine;phosphatidylinositol; sphingomyelin; phosphatidylethanolamine(cephalin); cardiolipin; phosphatidic acid; cerebrosides;dicetylphosphate; dipalmitoylphosphatidylglycerol; stearylamine;dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol ricinoleate;hexadecyl sterate; isopropyl myristate; tyloxapol; poly(ethyleneglycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. The surfactant component may be a mixture of differentsurfactants. These surfactants may be extracted and purified from anatural source or may be prepared synthetically in a laboratory. In apreferred embodiment, the surfactants are commercially available.

Those skilled in the art will recognize that this is an exemplary, notcomprehensive, list of substances with surfactant activity. Anysurfactant may be used in the production of AE nanoparticles.

Biologically Active Agents

Inventive AE nanoparticle compositions may be utilized to deliver one ormore biologically active agents. Thus, biologically active agents may bedelivered in conjunction with inventive AE nanoparticles. In someembodiments, the biologically active agent is included in the premix. Insome embodiments, the biologically active agent is added after AEnanoparticle formation.

Any biologically active agents, including, for example, therapeutic,diagnostic, prophylactic, nutritional, cosmetic, and/or dermatologicalagents, may be delivered according to the present invention. Suchbiologically active agents may be small molecules, organometalliccompounds, nucleic acids, proteins (including multimeric proteins,protein complexes, etc.), peptides, lipids, carbohydrates, herbs,hormones, metals, radioactive elements and compounds, drugs, vaccines,immunological agents, etc., and/or combinations thereof. Suchbiologically agents may be encapsulated within, adsorbed to the surfaceof, and/or present within the micellar membrane of inventive AEnanoparticles.

In some embodiments, the percent of biologically active agent in thepremix or in nanoparticles ranges from 0.1% to 25%. In some embodiments,the percentage of biologically active agent in the premix or innanoparticles ranges from 0.1% to 20%, from 0.1% to 15%, from 0.1% to10%, from 0.1% to 5%, or from 0.1% to 1%. In some embodiments, thepercentage of biologically active agent in the premix or innanoparticles ranges from 1% to 20%, from 5% to 20%, from 10% to 20%,from 15% to 20%, or from 15% to 25%. In some embodiments, the percentageof biologically active agent in the premix or in nanoparticles is lessthan 0.1%. In some embodiments, the percentage of biologically activeagent in the premix or in nanoparticles is greater than 25%. In someembodiments, the percentage of biologically active agent in the premixor in nanoparticles is approximately 0.1%, approximately 1%,approximately 2%, approximately 3%, approximately 4%, approximately 5%,approximately 6%, approximately 7%, approximately 8%, approximately 9%,approximately 10%, approximately 11%, approximately 12%, approximately13%, approximately 14%, approximately 15%, approximately 16%,approximately 17%, approximately 18%, approximately 19%, approximately20%, approximately 21%, approximately 22%, approximately 23%,approximately 24%, approximately 25%, or greater.

Relevant biologically active agents can be produced or obtainedaccording to any available method or approach. Biologically activeagents may contain, or be modified to contain, one or more moietiesintended to facilitate their use or delivery in conjunction withinventive nanoparticles. Such modification should not interfere with thebiological activity of the agent. In some embodiments, the modificationcan optionally be removed in vivo. For example, biologically activeagents may be detectably labeled and/or may be provided in a “pro” formthat is converted or modified after delivery into an active form.

In some embodiments, the biologically active agent is a small moleculeand/or organic compound with pharmaceutical activity. In someembodiments, the biologically active agent is a clinically-used drug. Insome embodiments, the drug is an antibiotic, anti-viral agent,anesthetic, anticoagulant, anti-cancer agent, inhibitor of an enzyme,steroidal agent, anti-inflammatory agent, anti-neoplastic agent,antigen, vaccine, antibody, decongestant, antihypertensive, sedative,birth control agent, progestational agent, anti-cholinergic, analgesic,anti-depressant, anti-psychotic, β-adrenergic blocking agent, diuretic,cardiovascular active agent, vasoactive agent, non-steroidalanti-inflammatory agent, etc.

The biologically active agents delivered may be a mixture ofpharmaceutically active agents. For example, a local anesthetic may bedelivered in combination with an anti-inflammatory agent such as asteroid. Local anesthetics may also be administered with vasoactiveagents such as epinephrine. To give but another example, an antibioticmay be combined with an inhibitor of the enzyme commonly produced bybacteria to inactivate the antibiotic (e.g., penicillin and clavulanicacid).

In some embodiments, the biologically active agent is a diagnosticagent. In some embodiments, diagnostic agents include gases;commercially available imaging agents used in positron emissionstomography (PET), computer assisted tomography (CAT), single photonemission computerized tomography, x-ray, fluoroscopy, and magneticresonance imaging (MRI); and contrast agents. Examples of suitablematerials for use as contrast agents in MRI include gadolinium chelates,as well as iron, magnesium, manganese, copper, and chromium. Examples ofmaterials useful for CAT and x-ray imaging include iodine-basedmaterials.

In some embodiments, the biologically active agent is a prophylacticagent. In some embodiments, prophylactic agents include vaccines.Vaccines may comprise isolated proteins or peptides, inactivatedorganisms and viruses, dead organisms and virus, genetically alteredorganisms or viruses, and cell extracts. Prophylactic agents may becombined with interleukins, interferon, cytokines, and adjuvants such ascholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents mayinclude antigens of such bacterial organisms as Streptococccuspnuemoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcuspyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillusanthraces, Clostridium tetani, Clostridium botulinum, Clostridiumperfringens, Neisseria meningitidis, Neisseria gonorrhoeae,Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi,Haemophilus parainjluenzae, Bordetella pertussis, Francisellatularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila,Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum,Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni,and the like; antigens of such viruses as smallpox, influenza A and B,respiratory syncytial virus, parainfluenza, measles, HIV,varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barrvirus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus,mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japaneseencephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, andE virus, and the like; antigens of fungal, protozoan, and parasiticorganisms such as Cryptococcus neoformans, Histoplasma capsulatum,Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsiaricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci,Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei,Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis,Schistosoma mansoni, and the like. These antigens may be in the form ofwhole killed organisms, peptides, proteins, glycoproteins,carbohydrates, or combinations thereof.

In some embodiments, the biologically active agent may be a protein. Asused herein, the terms “protein” and “peptide” can be usedinterchangeably. In certain embodiments, peptides range from about 5 to40, 10 to 35, 15 to 30, or 20 to 25 amino acids in size. Peptides frompanels of peptides comprising random sequences and/or sequences whichhave been varied consistently to provide a maximally diverse panel ofpeptides may be used.

In some embodiments, the biologically active agent may be an antibody.In some embodiments, antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric (i.e. “humanized”), single chain(recombinant) antibodies. In some embodiments, antibodies may havereduced effector functions and/or bispecific molecules. In someembodiments, antibodies may include Fab fragments and/or fragmentsproduced by a Fab expression library.

In some embodiments, the biologically active agent may be a nucleicacid. In some embodiments, the oligonucleotides comprise DNA, RNA,chimeric mixtures, derivatives, characteristic portions, and/or modifiedversions thereof. The oligonucleotides of the present invention may besingle-stranded and/or double-stranded. The oligonucleotide may bemodified at the base moiety, sugar moiety, and/or phosphate backbone,for example, to improve stability of the molecule, hybridization, etc.

In specific embodiments, a nucleic acid comprises an antisense moleculethat binds to a translational start site, transcriptional start site,and/or splice junctions. Antisense oligonucleotides will bind to atarget mRNA and/or prevent translation. Alternatively or additionally,the antisense oligonucleotide may bind to DNA of a target gene, such as,for example, a regulatory element.

In some embodiments, a nucleic acid comprises a ribozyme designed tocatalytically cleave target mRNA transcripts may be used to preventtranslation of a target mRNA and/or expression of a target (see, e.g.,PCT publication WO 90/11364; and Sarver et al., 1990, Science 247:1222;both of which are incorporated herein by reference).

Alternatively or additionally, endogenous target gene expression may bereduced by targeting deoxyribonucleotide sequences complementary to theregulatory region of the target gene (i.e., the target gene's promoterand/or enhancers) to form triple helical structures that preventtranscription of the target gene in target muscle cells in the body (seegenerally, Helene, 1991, Anticancer Drug Des. 6:569; Helene et al.,1992, Ann, N.Y. Acad. Sci. 660:27; and Maher, 1992, Bioassays 14:807;all of which are incorporated herein by reference).

In some embodiments, the biologically active agent is a nutraceuticalagent. In some embodiments, the nutraceutical agent provides basicnutritional value. In some embodiments, the nutraceutical agent provideshealth or medical benefits. In some embodiments, the nutraceutical agentis a dietary supplement.

In some embodiments, the nutraceutical agent is a vitamin. In someembodiments, the vitamin is one or more of vitamin A (retinoids),vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin),vitamin B5 (pantothenic acid), vitamin B6 (pyroxidone), vitamin B7(biotin), vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), vitaminC (ascorbic acid), vitamin D, vitamin E, or vitamin K.

In some embodiments, the nutraceutical agent is a mineral. In someembodiments, the mineral is one or more of bismuth, boron, calcium,chlorine, chromium, cobalt, copper, fluorine, iodine, iron, magnesium,manganese, molybdenum, nickel, phosphorus, potassium, rubidium,selenium, silicon, sodium, strontium, sulfur, tellurium, titanium,tungsten, vanadium, or zinc.

In some embodiments, the nutraceutical agent is an essential amino acid.In some embodiments, the amino acid is one or more of arginine,glutamine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan, or valine.

In some embodiments, nutraceutical agents may include fatty acids and/oromega-3 fatty acids (e.g. DHA or ARA), fruit and vegetable extracts,lutein, phosphatidylserine, lipoid acid, melatonin, glucosamine,chondroitin, aloe vera, guggul, green tea, lycopene, whole foods, foodadditives, herbs, phytonutrients, antioxidants, flavonoid constituentsof fruits, evening primrose oil, flaxseeds, fish and marine animal oils(e.g. cod liver oil), and probiotics. In some embodiments, nutraceuticalagents may include bio-engineered foods genetically-engineered to have adesired property (also known as “pharmafoods”).

Exemplary nutraceutical agents and dietary supplements are disclosed,for example, in Roberts et al., (Nutriceuticals: The CompleteEncyclopedia of Supplements, Herbs, Vitamins, and Healing Foods,American Nutriceutical Association, 2001; incorporated herein byreference). Nutraceutical agents and dietary supplements are alsodisclosed in Physicians' Desk Reference for Nutritional Supplements, 1stEd., 2001 and Physicians' Desk Reference for Herbal Medicines, 1st Ed.,2001 (incorporated herein by reference).

In some embodiments, AE nanoparticles loaded with nutraceutical agentscan be incorporated into food substances. For example, thenutraceutical-loaded AE nanoparticles can be dissolved into liquids,such as beverages.

In some embodiments, the biologically active agent is a cosmetic and/ordermatological agent. In some embodiments, the cosmetic and/ordermatological agent may optionally include excipients such assequestering agents, softeners, coloring materials (e.g. pigments anddyes), and fragrances. In some embodiments, the cosmetic and/ordermatological agent may be a composition including, but not limited to,skin softener, nutrition lotion, cleansing lotion, cleansing cream, skinmilk, emollient lotion, massage cream, emollient cream, make-up base,lipstick, facial pack or facial gel, cleaner formulation (e.g. shampoos,rinses, body cleanser, hair-tonics, and soaps), and dermatologicalcomposition (e.g. lotions, ointments, gels, creams, patches and sprays).

In some embodiments, the cosmetic and/or dermatological agent mayinclude vitamins and their derivatives (e.g. vitamin E and its esters,vitamin C and its esters, vitamins B, vitamin A alcohol or retinol andits esters), provitamins (e.g. panthenol, niacinamide orergocalciferol), antioxidants, phenolic compounds (e.g. benzoylperoxide), essential oils, humectants, sunscreen agents, moisturizingagents, proteins, ceramides, and pseudoceramides.

In some embodiments, the biologically active agent may be one or morebotulinum toxin peptides or protein complexes. In some embodiments, thebotulinum toxin may be one or more of botulinum toxin serotypes A, B,C₁, C₂, D, E, F, or G. In some embodiments, the botulinum toxin may bean isolated and/or purified botulinum toxin. In some embodiments, thebotulinum toxin may be a partially-isolated and/or partially-purifiedbotulinum toxin. In some embodiments, the botulinum toxin may be anative botulinum complex. In some embodiments, the botulinum toxin maybe associated with non-toxin proteins. In some embodiments, thebotulinum toxin may be a recombinantly-made botulinum toxin.

Those skilled in the art will recognize that this is an exemplary, notcomprehensive, list of biologically active agents. Any biologicallyactive agent may be encapsulated within or bound to the surface of AEnanoparticles.

In some embodiments, AE nanoparticles comprising a biologically activeagent may optionally include one or more release-retarding ingredientsto allow for controlled release of the agent. Any release-retardingingredient known in the art is suitable for use in making the inventiveAE nanoparticles. In some embodiments, release-retarding ingredients arehydrophilic and/or hydrophobic polymers. Release-retarding ingredientsinclude, for example celluloses or derivatives thereof, acrylicpolymers, ester polymers, vinyl-pyrrolidone-based polymers, gums, othernatural polymers, and/or combinations of these.

In some embodiments, the release-retarding ingredient is cellulose or aderivative thereof. In certain embodiments, the cellulose or derivativethereof comprises one or more of hydroxypropyl methylcellulose,methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose,hydroxypropyl ethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. In certain embodiments, the cellulose or derivative thereofis methylcellulose or a derivative thereof. In certain embodiments, thecellulose or derivative thereof is hydroxypropyl methylcellulose (HPMC).Those skilled in the art will appreciate that other cellulosic polymers,including other alkyl cellulosic polymers, can be utilized.

In some embodiments, the release-retarding ingredient is an acrylicpolymer. In certain embodiments, acrylic polymers include, for example,acrylic acid and methacrylic acid copolymers, methyl methacrylatecopolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate,aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylicacid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, the release-retarding ingredient is a polyester. Insome embodiments, polyesters include polyalkylene glycols,poly(glycolide-co-lactide), PEGylated poly(lactic-co-glycolic acid),poly(lactic acid), PEGylated poly(lactic acid), poly(glycolic acid),PEGylated poly(glycolic acid), co-polymers of polylactic andpolyglycolic acid, and derivatives thereof. In some embodiments,polyesters include, for example, polyanhydrides, poly(ortho ester)PEGylated poly(ortho ester), poly(caprolactone), PEGylatedpoly(caprolactone), polylysine, PEGylated polylysine, poly(ethyleneimine), PEGylated poly(ethylene imine), and derivatives thereof. In someembodiments, polyesters include, for example, polycaprolactone,poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, the release-retarding ingredient is a cross-linkedpolymer of poly(vinyl-pyrrolidone). In some embodiments, the polymer iscrosspovidone. In some embodiments, the polymer is un-cross-linkedpoly(vinyl-pyrrolidone). In some embodiments, the polymer is povidone.

In some embodiments, the release-retarding ingredient may be a naturalpolymer. In some embodiments, the natural polymer is a gum, including,for example, xanthan gum, alginic acid, caraya gum, sodium alginate,and/or locust bean gum. In some embodiments, the natural polymer may bea protein (e.g. albumin), lipid, nucleic acid, or carbohydrate.

Methods of Making AE Nanoparticles

In general, inventive nanoparticle compositions (e.g., botulinumnanoparticle compositions) may be prepared by any available method. Insome embodiments, nanoparticle compositions are prepared by chemicalmeans. However, chemical means often require toxic (typically organic)solvents; in some embodiments, nanoparticle compositions are prepared inaccordance with the present invention without utilizing such solvents.

In certain embodiments of the present invention, nanoparticlecompositions are prepared by preparing a premix and subjecting thepremix to high shear forces. As used herein, the term “shear force”refers to a force that is parallel to the face of a material, as opposedto a force that is perpendicular to the face of a material.

Any method known in the art can be used to generate high shear forces.According to the present invention, the use of mechanical energy (i.e.,high shear forces) can replace or minimize any requirement to use costlyand/or toxic chemical solvents; can increase the speed at whichnanoparticles assemble, can increase the yield of nanoparticlesgenerated in a particular mix of components, and/or can greatly reducethe overall cost of preparing nanoparticle compositions. Furthermore, inthose embodiments in which an agent such as a biologically active agent(e.g., botulinum toxin) is incorporated into inventive nanoparticlecompositions, the use of high shear force can increase the loadingcapacity of the nanoparticle as compared to traditional methods offorming nanoparticles. In traditional methods, loading of agents withinor on the surface of nanoparticles typically relies on diffusion of theagent to the interior and/or to the surface of the nanoparticle.According to the present invention, the use of high shear force canallow for the manufacture of smaller particles (e.g., on average) and/ora more narrow distribution of particle sizes in a nanoparticlecomposition.

In some embodiments, high shear forces are achieved by exposure to highpressure, for example by continuous turbulent flow at high pressure, forexample about 15,000 psi. In some embodiments, such high pressure iswithin the range of about 18,000 to about 26,000 psi; in someembodiments, it is within the range of about 20,000 to 25,000 psi. Insome embodiments, high shear forces are characterized by pressures of atleast 3,000 psi, 10,000 psi, 15,000 psi, 18,000 psi, 20,000 psi, 22,000psi, or 24,000 psi. In some embodiments, high shear forces arecharacterized by pressures of 16,000 psi, 17,000 psi, 18,000 psi, 19,000psi, 20,000 psi, 21,000 psi, 22,000 psi, 23,000 psi, 24,000 psi, or25,000 psi.

In some embodiments, cavitation is used to generate high shear forces.In some embodiments, high pressure homogenization is used to generatehigh shear forces.

In some embodiments, high shear force may be administered by passagethrough an instrument such as, for example, a Microfluidizer® Processor(Microfluidics Corporation/MFIC Corporation) or other like device.Microfluidizer® Processors provide high pressure and a resultant highshear rate by accelerating the product through microchannels to a highvelocity for size reduction to the nanoscale range. The fluid is splitin two and is pushed through microchannels with typical dimensions inthe order of 75 microns at high velocities (in the range of 50-300 m/s).As the fluid exits the microchannels it forms jets which collide withjets from opposing microchannels. In the channels the fluid experienceshigh shear (up to 10⁷ 1/s) which is orders of magnitude higher than thatof conventional technologies. Jet collisions result in mixing insubmicron level. Therefore, high shear and impact are responsible forparticle size reduction and mixing of multiphase fluids in theMicrofluidizer® technology.

In some embodiments of the present invention, a sample is“microfluidized” through exposure to high shear forces for a period oftime less than about 10 minutes. In some embodiments, the period of timeis less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute(s). In someembodiments, the period of time is within the range of about 1-2 minutesor less; in some embodiments, the period of time is about 30 seconds.

In some embodiments of the invention, a sample is “microfluidized”through a single exposure to high shear forces; such embodiments arereferred to herein as “single pass” microfluidization.

The present invention encompasses the recognition that subjecting apremix to high shear forces can generate a nanoparticle composition, andin particular can generate a uniform nanoparticle composition.

In some embodiments of the present invention, all of the componentspresent in the final nanoparticle composition are present in the premixand are subjected to high shear force to produce the nanoparticlecomposition. In some embodiments of the present invention, one or moreof the components that are present in the final nanoparticle compositionis/are missing from the premix or is/are present in the premix in asmaller amount than in the final nanoparticle composition. That is, insome embodiments of the present invention, one or more materials areadded to the nanoparticle composition after the premix is subjected tohigh shear force.

In certain embodiments of the invention, the premix is prepared as asolution prior to application of high shear force. In particular, fornanoparticle compositions that include at least one biologically activeagent (e.g., botulinum toxin), it is often desirable for thebiologically active agent to be dissolved in the premix before the highshear force is applied. Thus, in many embodiments, the biologicallyactive agent is soluble in at least one of the media (or in acombination of media utilized in the premix). In some embodiments of theinvention, such dissolution requires heating; in other embodiments itdoes not.

In some embodiments of the present invention, the premix components mayassemble into particles before the application of high shear force. Atleast some of such particles may be microparticles or evennanoparticles. In some embodiments, an inventive nanoparticlecomposition is prepared from a premix, wherein the premix is selectedfrom the group comprising a suspension or a microemulsion. In someembodiments, however, particle structures do not form in the premixbefore application of high shear force.

Methods of Use

In some embodiments, the present invention provides methods of using AEnanoparticles and/or nanoparticle compositions by delivering them(optionally in conjunction with a biologically active agent or othersubstance) to a subject. Such delivery may be via any route. Forexample, delivery may be orally, parenterally, intracisternally,intravaginally, subcutaneously, intraperitoneally, intramuscularly,intravenously, transdermally (topically), intradermally, bucally,rectally, and/or opthalmically.

In some embodiments, the invention provides methods of transdermallydelivering a biologically active agent to a subject by administering tothe subject one or more AE nanoparticles to the surface of the subject'sskin, wherein the biologically active agent is contained within or boundto the surface of the AE nanoparticles. In some embodiments, the subjectmay be a mammal (e.g. human).

In some embodiments, a composition for transdermal delivery of abiologically active agent may comprise AE nanoparticles containing theagent to be delivered. In some embodiments, the biologically activeagent may be encapsulated within the AE nanoparticles. In someembodiments, the biologically active agent may be bound to the surfaceof the AE nanoparticles.

Traditionally, attempts at transdermal administration of substances haverequired a step of improving the permeability of the skin before thesubstance is applied. Some attempts have included using chemicalpenetration enhancing agents that act on the skin's surface to increasethe permeability of substances through the skin. The use of thesechemical penetration enhancing agents is often painful and may damagethe surface of the skin. Other attempts have included the use ofultrasound or iontophoresis or other forms of energy to facilitate thepermeation of substances through the skin as well as micro-puncture orhigh-energy techniques to create micro-channels across the surfacelayers of the skin, such as the stratum corneum. The AE nanoparticles ofthe present invention can achieve transdermal delivery of a biologicallyactive agent without requiring the use of abrasive or otherskin-disrupting agents (whether chemical, mechanical, electrical,magnetic, etc.).

In some embodiments, a composition for transdermal delivery of acomposition comprising AE nanoparticles for transdermal delivery of abiologically active agent may be in the form of a cosmetic formulationincluding, but not limited to, a skin softener, nutrition lotion typeemulsion, cleansing lotion, cleansing cream, skin milk, emollientlotion, massage cream, emollient cream, make-up base, lipstick, facialpack or facial gel, cleaner formulation (e.g. shampoos, rinses, bodycleanser, hair-tonics, or soaps), and dermatological composition (e.g.lotions, ointments, gels, creams, patches or sprays).

In some embodiments, a composition for transdermal delivery of abiologically active agent may be in the form of a transdermal patch. Theuse of adhesive patches is well known in the art (for example, see U.S.Pat. Nos. 296,006 (design); 6,010,715; 5,591,767; 5,008,110; 5,683,712;5,948,433; and 5,965,154. In some embodiments, the transdermal patch maycomprise an adhesive layer, which may be applied to a person's skin. Insome embodiments, the transdermal patch may comprise a depot orreservoir for holding a biologically active agent or composition. Insome embodiments, the transdermal patch comprises an exterior surfacethat may prevent leakage of the agent or composition from the depot. Insome embodiments, the exterior surface of a patch may be non-adhesive.

In some embodiments, the composition comprising AE nanoparticles fortransdermal delivery of a biologically active agent may be incorporatedinto a patch so that the AE nanoparticles remain stable for extendedperiods of time. The AE nanoparticles may be incorporated into apolymeric matrix that stabilizes the AE nanoparticles and permits the AEnanoparticles to diffuse from the matrix and from the patch. In someembodiments, the AE nanoparticles may be incorporated into the adhesivelayer of the patch. In one embodiment, the adhesive layer may beheat-activated. In certain embodiments, temperatures of about 37° C. maycause the adhesive to slowly liquefy so that the AE nanoparticlesdiffuse through the skin. In certain embodiments, the adhesive mayremain tacky when stored at less than 37° C. In some embodiments, theadhesive loses its tackiness as it liquefies at temperatures of about37° C. In some embodiments, the administration of the AE nanoparticlesis complete once the patch no longer adheres to the skin.

In some embodiments, the compositions comprising AE nanoparticles fortransdermal delivery of a biologically active agent may be used in anapplication device that permits application of the composition to atarget site on the skin without applying the composition to non-targetsite areas of the skin. In some embodiments, a device may be employedthat allows the composition to be applied without first applying thecomposition to one's fingers, which may lead to undesirable paralysis ofthe fingers. Suitable devices include spatulas, swabs, syringes withoutneedles, and adhesive patches. Use of spatulas or swabs, or the like mayrequire the device to be inserted into a container containing thecomposition. Using syringes or adhesive patches may be accomplished byfilling the syringe or patch with the composition. The composition maybe topically spread by the spatulas or swabs, or may be expelled fromthe syringes onto the person's skin.

In some embodiments, the biologically active agent may be one or morebotulinum toxin peptides or protein complexes. In some embodiments, thebotulinum toxin may be one or more of botulinum toxin serotypes A, B,C₁, C₂, D, E, F, or G. In some embodiments, the botulinum toxin may bean isolated and/or purified botulinum toxin. In some embodiments, thebotulinum toxin may be a partially-isolated and/or partially-purifiedbotulinum toxin. In some embodiments, the botulinum toxin may be anative botulinum complex. In some embodiments, the botulinum toxin maybe associated with non-toxin proteins. In some embodiments, thebotulinum toxin may be a recombinantly-made botulinum toxin.

In some embodiments, the botulinum toxin within a composition fortransdermal delivery may be present in an amount so that between about10⁻³ U/kg and 10 U/kg pass through a patient's skin. In someembodiments, the botulinum toxin may be present in an amount so thatbetween about 10⁻² U/kg and about 1 U/kg pass through the patient'sskin. In some embodiments, the botulinum toxin may be present in anamount so that between about 10⁻¹ U/kg and about 1 U/kg pass through thepatient's skin. In some embodiments, the botulinum toxin may be presentin an amount so that between about 0.1 U and about 5 U pass through thepatient's skin. As used herein, “Units” (“U”) are biologicallyequivalent or bioactively equivalent to Units defined by commercialmanufacturers of botulinum toxin.

In one embodiment, dosages of botulinum toxin can range from as low asabout 1 U to as high as about 20,000 U. The particular dosages may varydepending on the condition being treated and therapeutic regime beingutilized. For example, treatment of subdermal, hyperactive muscles mayrequire high transdermal dosages (for example, 200 U to 20,000 U) ofbotulinum toxin. In comparison, treatment of neurogenic inflammation orhyperactive sweat glands may require relatively small transdermaldosages (for example, about 1 U to about 1,000 U) of botulinum toxin. Insome embodiments, the composition may comprise an amount of botulinumtoxin sufficient to achieve a therapeutic effect lasting between 1 monthand 5 years. In some embodiments, the composition comprising botulinumtoxin may be formulated to avoid potential complications including, butnot limited to, systemic toxicity or botulism poisoning.

In some embodiments, the present invention provides methods of treatingfacial wrinkles. In some embodiments, nanoparticle compositionscomprising AE nanoparticles for the transdermal delivery of abiologically active agent may be used to treat facial wrinkles. In someembodiments, nanoparticle compositions comprising AE nanoparticles forthe transdermal delivery of botulinum toxin may be used to treat facialwrinkles. In some embodiments, facial wrinkles may include glabellarwrinkles, facial lines (e.g. hyperkinetic facial lines), forehead frownlines, midfacial wrinkles, mouth wrinkles, neck lines and banding (e.g.platysma bands), and chin creases.

In some embodiments, the present invention provides methods of treatingneuromuscular disorders and conditions in a subject. In someembodiments, nanoparticle compositions comprising AE nanoparticles forthe transdermal delivery of a biologically active agent may be used totreat neuromuscular disorders and conditions involving muscular spasmand/or contracture. In some embodiments, nanoparticle compositionscomprising AE nanoparticles for the transdermal delivery of botulinumtoxin may be used to treat neuromuscular disorders and conditions. Insome embodiments, neuromuscular disorders and conditions involvingmuscular spasm and/or contracture include, but are not limited to,various forms of palsy, facial contracture, dystonia, hemifacial spasm,tremor, spasticity (e.g. resulting from multiple sclerosis),retroorbital muscle, and various other ophthalmologic conditions(Carruthers et al., 1996, J Am. Acad. Dermatol., 34:788; incorporatedherein by reference). In some embodiments, the present invention doesnot provide methods of treating neuromuscular disorders and conditionsinvolving muscular spasm and/or contracture in a subject.

In some embodiments, the present invention provides methods of treatinghyperhidrosis (i.e., a medical condition in which a person sweatsexcessively and unpredictably) in a subject. In some embodiments,nanoparticle compositions comprising AE nanoparticles for thetransdermal delivery of a biologically active agent may be used to treathyperhidrosis. In some embodiments, nanoparticle compositions comprisingAE nanoparticles for the transdermal delivery of botulinum toxin may beused to treat hyperhidrosis. In some embodiments, the present inventiondoes not provide methods of treating hyperhidrosis in a subject.

In some embodiments, the present invention provides methods of treatingheadache in a subject. In some embodiments, nanoparticle compositionscomprising AE nanoparticles for the transdermal delivery of abiologically active agent may be used to treat headache. In someembodiments, nanoparticle compositions comprising AE nanoparticles forthe transdermal delivery of botulinum toxin may be used to treatheadache. In some embodiments, the present invention does not providemethods of treating headache in a subject.

In some embodiments, the present invention provides methods of treatingprostate hyperplasia in a subject. In some embodiments, nanoparticlecompositions comprising AE nanoparticles for the transdermal delivery ofa biologically active agent may be used to treat prostate hyperplasia.In some embodiments, nanoparticle compositions comprising AEnanoparticles for the transdermal delivery of botulinum toxin may beused to treat prostate hyperplasia. In some embodiments, the presentinvention does not provide methods of treating prostate hyperplasia in asubject.

In some embodiments, the present invention provides a method of imaginga disorder (e.g. cancer) in a subject by labeling one or more AEnanoparticles with a reporter group and with a targeting agent thatbinds to a target associated with the disorder; administering thelabeled particles to the subject under conditions and in an amountsufficient to bind to the target; and imaging the reporter group,thereby imaging the disorder.

In some embodiments, the inventive AE nanoparticles are used to deliverdrugs to a subject. In some embodiments, nanoparticle compositions maycomprise AE nanoparticles which contain drugs, including but not limitedto, antibiotics, anti-viral agents, anesthetics, anticoagulants,anti-cancer agents, inhibitors of enzymes, steroidal agents,anti-inflammatory agents, anti-neoplastic agents, antigens, vaccines,antibodies, decongestants, antihypertensives, sedatives, birth controlagents, progestational agents, anti-cholinergics, analgesics,anti-depressants, anti-psychotics, β-adrenergic blocking agents,diuretics, cardiovascular active agents, vasoactive agents, hormones(e.g. insulin, estradiol), and non-steroidal anti-inflammatory agents.

Pharmaceutical Compositions

The present invention provides AE nanoparticles. In some embodiments,the present invention provides for pharmaceutical compositionscomprising AE nanoparticles, as described herein. The present inventionprovides pharmaceutical compositions comprising AE nanoparticlescontaining a therapeutically effective amount of a biologically activeagent. Such pharmaceutical compositions may optionally comprise one ormore additional therapeutically-active substances. In accordance withone embodiment, a method of administering a pharmaceutical compositioncomprising AE nanoparticles containing a therapeutically effectiveamount of a therapeutic agent to a patient in need thereof. In someembodiments, the compositions are administered to humans.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and/or other primates; mammals, includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and/or dogs; and/or birds, including commercially relevant birds such aschickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject and/or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

It will also be appreciated that certain of the compounds of the presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable salts, esters, saltsof such esters, or any other adduct or derivative which uponadministration to a patient in need is capable of providing, directly orindirectly, a compound as otherwise described herein, or a metabolite orresidue thereof, e.g., a prodrug.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describe pharmaceutically acceptable salts in detail (1977, J.Pharm. Sci., 66:1; incorporated herein by reference). Salts can beprepared in situ during the final isolation and purification of thecompounds of the invention, or separately by reacting the free basefunctionality with a suitable organic or inorganic acid. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid, or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate, and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters which hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moiety advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates. In certainembodiments, the esters are cleaved by enzymes such as esterases.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of the above formula, for example by hydrolysis in blood. Athorough discussion is provided in T. Higuchi and V. Stella, Pro-drugsas Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series and inE. B. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987 (both of which areincorporated herein by reference).

As described above, the pharmaceutical formulations of the presentinvention may additionally comprise a pharmaceutically acceptableexcipient, which, as used herein, includes any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington's TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,(Lippincott, Williams & Wilkins, Baltimore, Md., 2006) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, theexcipient is approved for use in humans and for veterinary use. In someembodiments, the excipient is approved by United States Food and DrugAdministration. In some embodiments, the excipient is pharmaceuticalgrade. In some embodiments, the excipient meets the standards of theUnited States Pharmacopoeia (USP), the European Pharmacopoeia (EP), theBritish Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the inventive formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and perfuming agents can also bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and combinations thereof

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminumsilicate]), long chain amino acid derivatives, high molecular weightalcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60],polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate[Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span65], glyceryl monooleate, sorbitan monooleate [Span 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben,Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certainembodiments, the preservative is an anti-oxidant. In other embodiments,the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., andcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

The pharmaceutical compositions may be administered to animals,preferably mammals (e.g., domesticated animals, cats, dogs, mice, rats),and more preferably humans. Any method of administration may be used todeliver the pharmaceutical compositions to the animal. In certainembodiments, the pharmaceutical composition is administered orally. Inother embodiments, the pharmaceutical composition is administeredparenterally.

In some embodiments of the invention, a method for the treatment offacial wrinkles is provided comprising administering a therapeuticallyeffective amount of AE nanoparticles comprising botulinum toxin to asubject in need thereof, in such amounts and for such time as isnecessary to achieve the desired result. In certain embodiments of thepresent invention a “therapeutically effective amount” of the AEnanoparticles comprising botulinum toxin is that amount effective fortreating facial wrinkles including, but not limited to, glabellarwrinkles, facial lines (e.g. hyperkinetic facial lines), forehead frownlines, midfacial wrinkles, mouth wrinkles, neck lines and banding (e.g.platysma bands), and chin creases.

In some embodiments of the invention, a method for the treatment ofhyperhidrosis is provided comprising administering a therapeuticallyeffective amount of AE nanoparticles comprising botulinum toxin to thehands, feet, and/or underarms of a subject in need thereof, in suchamounts and for such time as is necessary to achieve the desired result.

The compositions, according to the method of the present invention, maybe administered using any amount and any route of administrationeffective for treating facial wrinkles. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the infection, the particularcomposition, its mode of administration, its mode of activity, and thelike. The compositions of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of thecompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient ororganism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

The pharmaceutical compositions of the present invention may beadministered by any route. In some embodiments, the pharmaceuticalcompositions of the present invention are administered variety ofroutes, including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(as by powders, ointments, creams, and/or drops), mucosal, bucal,enteral, sublingual, and/or as an oral spray, nasal spray, and/oraerosol. In general the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),the condition of the patient (e.g., whether the patient is able totolerate oral administration), etc. At present the oral and/or nasalspray and/or aerosol route is most commonly used to deliver therapeuticagents directly to the lungs and/or respiratory system. However, theinvention encompasses the delivery of the inventive pharmaceuticalcomposition by any appropriate route taking into consideration likelyadvances in the sciences of drug delivery.

In certain embodiments, the compounds of the invention may beadministered orally or parenterally at dosage levels sufficient todeliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kgto about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg,preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kgto about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and morepreferably from about 1 mg/kg to about 25 mg/kg, of subject body weightper day, one or more times a day, to obtain the desired therapeuticeffect. The desired dosage may be delivered three times a day, two timesa day, once a day, every other day, every third day, every week, everytwo weeks, every three weeks, or every four weeks. In certainembodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations).

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active compounds, the liquid dosage forms may contain inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents. In certainembodiments for parenteral administration, the compounds of theinvention are mixed with solubilizing agents such an Cremophor,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugar as wellas high molecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compoundof this invention may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, theactive component is admixed under sterile conditions with apharmaceutically acceptable carrier and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, the rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662 (all of which areincorporated herein by reference). Intradermal compositions may beadministered by devices which limit the effective penetration length ofa needle into the skin, such as those described in PCT publication WO99/34850 (incorporated herein by reference) and functional equivalentsthereof. Jet injection devices which deliver liquid vaccines to thedermis via a liquid jet injector and/or via a needle which pierces thestratum corneum and produces a jet which reaches the dermis aresuitable. Jet injection devices are described, for example, in U.S. Pat.Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537; all ofwhich are incorporated herein by reference. Ballistic powder/particledelivery devices which use compressed gas to accelerate vaccine inpowder form through the outer layers of the skin to the dermis aresuitable. Alternatively or additionally, conventional syringes may beused in the classical mantoux method of intradermal administration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65.degree. F. at atmospheric pressure. Generallythe propellant may constitute 50 to 99.9% (w/w) of the composition, andthe active ingredient may constitute 0.1 to 20% (w/w) of thecomposition. The propellant may further comprise additional ingredientssuch as a liquid non-ionic and/or solid anionic surfactant and/or asolid diluent (which may have a particle size of the same order asparticles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may provide the active ingredient in the form of droplets of asolution and/or suspension. Such formulations may be prepared, packaged,and/or sold as aqueous and/or dilute alcoholic solutions and/orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization and/oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration may have an averagediameter in the range from about 0.1 nm to about 200 nm.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical composition ofthe invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the active ingredient andhaving an average particle from about 0.2 μm to 500 μm. Such aformulation is administered in the manner in which snuff is taken, i.e.by rapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition of theinvention may be prepared, packaged, and/or sold in a formulationsuitable for buccal administration. Such formulations may, for example,be in the form of tablets and/or lozenges made using conventionalmethods, and may, for example, 0.1% to 20% (w/w) active ingredient, thebalance comprising an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising the active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of the additional ingredientsdescribed herein.

A pharmaceutical composition of the invention may be prepared, packaged,and/or sold in a formulation suitable for ophthalmic administration.Such formulations may, for example, be in the form of eye dropsincluding, for example, a 0.1%/1.0% (w/w) solution and/or suspension ofthe active ingredient in an aqueous or oily liquid carrier. Such dropsmay further comprise buffering agents, salts, and/or one or more otherof the additional ingredients described herein. Otheropthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation. Ear drops and/or eye drops are contemplated asbeing within the scope of this invention.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be employed in combinationtherapies. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another anticancer agent), or they may achievedifferent effects (e.g., control of any adverse effects).

The pharmaceutical compositions of the present invention may beadministered either alone or in combination with one or more othertherapeutic agents. By “in combination with,” it is not intended toimply that the agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. The compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent. Additionally, the invention encompasses the delivery of theinventive pharmaceutical compositions in combination with agents thatmay improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

The particular combination of therapies (therapeutics and/or procedures)to employ in a combination regimen will take into account compatibilityof the desired therapeutics and/or procedures and/or the desiredtherapeutic effect to be achieved. It will be appreciated that thetherapies employed may achieve a desired effect for the same disorder(for example, an inventive compound may be administered concurrentlywith another agent used to treat the same disorder), and/or they mayachieve different effects (e.g., control of any adverse effects).

The pharmaceutical compositions of the present invention may beadministered alone and/or in combination with other agents that are usedto treat the symptoms of facial wrinkles. To give but a few examples,the pharmaceutical compositions of the present invention could beadministered in combination with agents such as retinoic acid, vitamin Cand/or E, and/or hyaluronic acid, pentapeptides (e.g.,lys-thr-thr-lys-ser), and/or hexapeptides (e.g. acetyl hexapeptide-3,also known as Argireline), depending on the route of administration.Novel compositions comprising pentapeptides and hexapeptides are furtherdescribed in PCT application serial number PCT/US07/086040 entitled“Peptide Nanoparticles and Uses Thereof,” filed on Nov. 30, 2007.

In will further be appreciated that therapeutically active agentsutilized in combination may be administered together in a singlecomposition or administered separately in different compositions.

In general, it is expected that agents utilized in combination with beutilized at levels that do not exceed the levels at which they areutilized individually. In some embodiments, the levels utilized incombination will be lower than those utilized individually.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

Kits

In some embodiments, the present invention relates to a kit forconveniently and/or effectively carrying out the methods in accordancewith the present invention. In general, an inventive pharmaceutical packand/or kit comprises one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Such kits are especially suited for the delivery of solid oral formssuch as tablets and/or capsules. In some embodiments, such a kitincludes a number of unit dosages, and may include a card having thedosages oriented in the order of their intended use. A memory aid may beprovided, for example in the form of numbers, letters, and/or othermarkings and/or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.Alternatively, placebo dosages, and/or calcium dietary supplements,either in a form similar to or distinct from the dosages of thepharmaceutical compositions, may be included to provide a kit in which adosage is taken every day. Optionally associated with such container(s)may be a notice in the form prescribed by a governmental agencyregulating the manufacture, use and/or sale of pharmaceutical products,which notice reflects approval by the agency of manufacture, use and/orsale for human administration.

The invention provides a kit comprising AE nanoparticles of theinvention and/or instructional materials which describe administeringthe inventive AE nanoparticles to a cell and/or a tissue of a subject.In another embodiment, a kit may comprise a dispersion medium suitablefor dissolving and/or suspending the inventive AE nanoparticles prior toadministering the compound to the subject.

EXEMPLIFICATION

The following examples are only intended to provide illustrations ofspecific embodiments contemplated by the present invention. The examplesare not intended in any way to be limiting.

Example 1: Formulation of a Self-Assembling Pullulan andPolycaprolactone Nanosphere

A mixture of 2.5 g of soybean oil and 2.5 g polysorbate 80 (Tween-80)was prepared. The mixture was stirred and heated at 40° C. for 5minutes. 50 ml deionized water was added, and the resulting mixture wasstirred and heated at 40° C. for 10 minutes. 5 ml DMSO containing 0.905g pullulan and polycaprolactone was added, and the resulting mixture wasstirred and heated at 45° C. for 10 minutes. 5 ml was taken for apre-process sample. The remaining mixture was microfluidized in a singlepass at 24,000 psi. The particle size of the pre-process samplewas >4000 nm. The particle size after microfluidization was 155 nm.

Example 2: Sample Preparation for Microfluidized Sample (Per Sample)

A mixture of 100 μl of microfluidized sample and 900 μl of reagent (0.1M sodium phosphate buffer, 1 mM EDTA, 0.25% Triton X-100, 160 IU/mL oftriglyceride hydrolase, and 1 IU/ml of cholesterol esterase) wasprepared in a 8 ml glass vial.

The resulting mixture was incubated at ambient temperature in the darkfor 1 hour. 100 μl of 5% sodium dodecyl sulfate was added, and theresulting mixture was vortexed for 30 seconds. 1 ml of ethanol wasadded, and the resulting mixture was vortexed for 30 seconds. 100 μl ofan internal standard was added, and the resulting mixture was vortexedfor 30 seconds. 4 ml of a 1:1 mixture of hexane:ether with 1% ethanoland 0.1% BHT was added. Ethanol and BHT stabilize the ether to preventperoxide formation. The resulting mixture was vortexed for 60 secondsthen centrifuged for 2 minutes on medium speed. The supernatant wasextracted with a glass pipet and was stored at −80° C. for up to 30days. The supernatant was evaporated and redissolved in 40 μl ofmethanol. 30 μl was injected into an high-pressure liquid chromatography(HPLC) apparatus.

Example 3: Botulinum Toxin A Formulation with Pullulan andPolycaprolactone

A mixture of 1.6 g of soybean oil and 1.6 g of polysorbate 80 (Tween-80)is prepared and stirred for five minutes. In a separate container, amixture of 100 ng of botulinum toxin A and 20 ml 0.9% saline is preparedand stirred for five minutes. The mixture of saline and botulinum toxinA is added to the mixture of oil and Tween-80 and stirred for 10minutes. 5 ml of DMSO containing 0.905 grams pullulan andpolycaprolactone are added, and the resulting mixture is stirred for 10minutes. A 5 ml pre-process sample is taken. The remaining mixture ismicrofluidized in a single pass at 24,000 psi. The particle size beforeand after microfluidization is measured.

Example 4: Vitamin E Formulation with Pullulan and Polycaprolactone

A mixture of 2.5 g soybean oil and 1 g vitamin E is prepared. 2.5 gpolysorbate 80 (Tween-80) is added. The resulting mixture is stirred andheated at 40° C. for 5 minutes. 50 ml water is added to the mixture, andthe resulting mixture is stirred and heated at 40° C. for 10 minutes. 5ml DMSO containing 0.905 g pullulan and polycaprolactone is added. Theresulting mixture is stirred and heated at 45° C. for 10 minutes. A 5 mlpre-process sample is taken. The remaining mixture is microfluidized ina single pass at 24,000 psi. The particle size before and aftermicrofluidization is measured.

Example 5: Tocopherol (Vitamin E) Analysis

Delta tocopherol concentrations can be measured for a patient bloodplasma or for nanoparticle compositions. Delta tocopherol concentrationsare determined by adding 200 μL of plasma or nanoparticle compositionwith 10 μL of retinyl acetate (internal standard; 10 μg/mL) and 200 μLof ethanol containing butylated hydroxytoluene (BHT) (10 mg/L) and 1.0mL hexane followed by vortex mixing. The samples are centrifuged at500×g for 5 minutes and the organic layer transferred to fresh tube. Thesample residues are re-extracted with 1.0 mL of hexane and the organiclayers are combined. The organic layers are evaporated under N₂ andreconstituted with 200 μL of ethanol containing BHT (10 mg/dL) andinjected into an HPLC. The HPLC system is a Model 5600 CoulArray8-channel system with two Model 580 pumps, a high-pressure gradientmixer, a peek pulse damper, a Model 540 autoinjector, a CoulArrayThermostatic Chamber and a serial array of eight coulometric electrodes(ESA Laboratories, Inc., Chelmsford, Mass., USA). The column is a3.0×150 mm, 3 μM, Supelcosil LC-18 (Supelco, Bellefonte, Pa., USA). Themobile phase consists of methanol/1Propanol/1 M ammonium acetate(78:20:2 v:v:v) at a flow rate of 0.8 mL/min. The concentrations ofdelta tocopherol are determined by external standardization usingpurified solutions of delta tocopherol standards (Sigma Chemicals, St.Louis, Mo., USA).

EQUIVALENTS AND SCOPE

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Those of ordinary skill in the art will appreciate that variouschanges and modifications to this description may be made withoutdeparting from the spirit or scope of the present invention, as definedin the following claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims or from relevant portions of thedescription is introduced into another claim. For example, any claimthat is dependent on another claim can be modified to include one ormore limitations found in any other claim that is dependent on the samebase claim. Furthermore, where the claims recite a composition, it is tobe understood that methods of using the composition for any of thepurposes disclosed herein are included, and methods of making thecomposition according to any of the methods of making disclosed hereinor other methods known in the art are included, unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. For example, itis to be understood that any of the compositions of the invention can beused for inhibiting the formation, progression, and/or recurrence ofadhesions at any of the locations, and/or due to any of the causesdiscussed herein or known in the art. It is also to be understood thatany of the compositions made according to the methods for preparingcompositions disclosed herein can be used for inhibiting the formation,progression, and/or recurrence of adhesions at any of the locations,and/or due to any of the causes discussed herein or known in the art. Inaddition, the invention encompasses compositions made according to anyof the methods for preparing compositions disclosed herein.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Any embodiment, element, feature, application, or aspect ofthe compositions and/or methods of the invention (e.g., any amphiphilicentity, any component of an amphiphilic entity, any polymer, anybiologically active agent, any surfactant, any dispersion medium, anyrelease-retarding ingredient, any AE nanoparticle or compositioncomprising any AE nanoparticle, any route or location of administration,any purpose for which a composition is administered, etc.), can beexcluded from any one or more claims. For purposes of brevity, all ofthe embodiments in which one or more elements, features, purposes, oraspects are excluded are not set forth explicitly herein.

1. A nanoparticle composition comprising a population of particles in adispersion medium and a botulinum toxin, wherein the nanoemulsioncomprises oily particles dispersed within an aqueous dispersion medium,or wherein the nanoemulsion comprises aqueous particles dispersed withinan oily dispersion medium, wherein the majority of particles havediameters between 10 and 300 nanometers, and wherein the particlescomprise hyaluronic acid, and wherein the nanoemulsion penetrates theskin of the subject without changing or altering the structure of theskin. 2.-11. (canceled)
 12. The nanoparticle composition of claim 1,wherein fewer than 5% of the particles have a diameter in excess of 300nm. 13.-25. (canceled)
 26. The nanoparticle composition of claim 1,wherein the difference between the minimum particle diameter and themaximum particle diameter does not exceed approximately 600 nm. 27.-28.(canceled)
 29. The nanoparticle composition of claim 1, wherein thedifference between the minimum particle diameter and the maximumparticle diameter does not exceed approximately 300 nm.
 30. (canceled)31. The nanoparticle composition of claim 1, wherein the differencebetween the minimum particle diameter and the maximum particle diameterdoes not exceed approximately 100 nm. 32.-39. (canceled)
 40. Thenanoparticle composition of claim 1, wherein the particles have anaverage diameter ranging between 50-250 nm. 41.-67. (canceled)
 68. Thenanoparticle composition of claim 1, wherein the nanoparticlecomposition was generated by microfluidization. 69.-72. (canceled) 73.The nanoparticle composition of claim 68, wherein the microfluidizationis single-pass microfluidization. 74.-78. (canceled)
 79. Thenanoparticle composition of claim 78, wherein the botulinum toxin (a) isencapsulated within the particles; (b) is nestled within the particlemembrane; (c) is associated with the particle surface; or a combinationthereof. 80.-96. (canceled)
 97. The nanoparticle composition of claim 1,wherein the nanoparticle composition penetrate the top layer of skin ofthe subject without the use of skin permeation enhancers or abrasives.98.-116. (canceled)
 117. The nanoparticle composition of claim 1,wherein the nanoparticle composition comprises an oil and a surfactant.118. The nanoparticle composition of claim 117, wherein the oil andsurfactant are present in a ratio ranging from 0.5-2.0. 119.-122.(canceled)
 123. The nanoparticle composition of claim 117, wherein thepercent of oil in the nanoparticle composition ranges from 1%-30%.124.-186. (canceled)
 187. The nanoparticle composition of claim 1,wherein the majority of particles have diameters below a specified sizeand within a specified range.
 188. The nanoparticle composition of claim1, wherein more than 70% of the majority of particles have diametersbelow a specified size and within a specified range.
 189. Thenanoparticle composition of claim 1, wherein more than 90% of themajority of particles have diameters below a specified size and within aspecified range.
 190. The nanoparticle composition of claim 1, whereinmore than 99.5% of the majority of particles have diameters below aspecified size and within a specified range.
 191. The nanoparticlecomposition of claim 1, wherein more than 99.9% of the majority ofparticles have diameters below a specified size and within a specifiedrange.
 192. The nanoparticle composition of claim 1, which has a zetapotential ranging between −25 mV to +25 mV.
 193. A method for treating asubject having a dermatological condition, the method comprisingtransdermally administering to the subject the nanoparticle compositionof claim 1.